CN110050063B - Acid alpha-glucosidase variants and their uses - Google Patents

Abstract

The present invention relates to variants of acid alpha-glucosidases and their uses.

Description

Acid alpha-glucosidase variants and their uses

The present invention relates to variants of acid alpha-glucosidase (GAA) and their uses.

Pompe disease, also known as glycogen storage disease type II (GSD) and acid maltase deficiency, is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) Autosomal recessive metabolic myopathies. GAA is an exo-1,4 and 1,6-alpha-glucosidase that hydrolyzes glycogen to glucose in lysosomes. GAA deficiency leads to glycogen accumulation in lysosomes and causes progressive damage to respiratory, cardiac, and skeletal muscles. The disease ranges from a rapidly developing course in infancy that is usually fatal before 1-2 years of age, to a more slowly developing heterogeneous process causing significant morbidity and early death in children and adults. Hirschhorn RR, The Metabolic and Molecular Bases of Inherited Disease, 3:3389-3420 (2001, McGraw-Hill); Vander Ploeg and Reuser, Lancet 372:1342-1351 (2008).

Current human therapies for the treatment of Pompe disease include the administration of recombinant human GAA, also known as enzyme replacement therapy (ERT). ERT has been shown to be effective in severe GSD II in infancy. However, the benefits of enzyme therapy are limited by the need for frequent infusions and the production of inhibitory antibodies against recombinant hGAA (Amalfitano, A. et al., (2001) Genet. In Med. 3:132-138). Furthermore, ERT is not efficient in correcting the entire body, possibly due to a combination of poor biodistribution of the protein after peripheral intravenous delivery, insufficient uptake by several tissues, and high immunogenicity.

As an alternative or adjunct to ERT, the feasibility of gene therapy for GSD-II has been studied. (Amalfitano, A. et al., (1999) Proc. Natl. Acad. Sci. USA 96: 8861-8866; Ding, E. et al., (2002) Mol. Ther. 5: 436-446; Fraites, T. J. et al., (2002) ) Mol. Ther. 5:571-578; Tsujino, S. et al. (1998) Hum. Gene Ther. 9: 1609-1616). However, muscle-directed gene transfer to correct genetic defects must face limitations due to the systemic nature of the disease and the fact that muscle expression of the transferred gene tends to be more immunogenic compared with other tissues.

Doerfler et al., 2016 described the combined administration of two constructs encoding human codon-optimized GAA, one under the control of a liver-specific promoter and the other under the control of a muscle-specific promoter. Down. Liver-specific promoter-driven GAA expression was used to promote immune tolerance to GAA in a Gaa ^−/− mouse model, whereas muscle-specific promoter-driven GAA expression was provided in a subset of tissues targeted by the therapy Expression of therapeutic proteins. However, this strategy is not entirely satisfactory as it requires the use of multiple constructs and it does not result in systemic expression of GAA.

The use of modified GAA proteins has been proposed in the past to improve lysosomal storage disease treatment. Specifically, application WO2004064750 and Sun et al., 2006, disclose a chimeric GAA polypeptide containing a signal peptide operably linked to GAA as a way to increase targeting of the protein to the secretory pathway.

However, the therapies available to patients are not entirely satisfactory, and there is still a need in the art for improved GAA polypeptides and GAA production. Specifically, there remains a need for long-term efficacy of treatments using GAA, high levels of GAA production, improved immune tolerance to the produced GAA polypeptides, and increased uptake of GAA by cells and tissues that require it. Furthermore, in WO2004064750 and Sun et al., 2006, the tissue distribution of the chimeric GAA polypeptides disclosed therein is not entirely satisfactory. Therefore, there remains a need for comprehensive therapeutic GAA polypeptides that allow correction of glycogen accumulation in most, if not all, target tissues.

Contents of the invention

The present invention relates to GAA variants that are expressed and secreted at higher levels than wild-type GAA protein, triggering improved correction of systemic glycogen pathological accumulation, and causing the induction of immune tolerance to GAA .

According to one aspect, the invention relates to a truncated GAA polypeptide comprising a deletion of at least one amino acid from the N-terminal terminus of a parent GAA polypeptide, wherein said parent polypeptide corresponds to a precursor form of a GAA polypeptide that does not contain a signal peptide. In a specific embodiment, the truncated GAA polypeptide has at least 2, especially at least 2, especially at least 3, especially at least 4, deleted at its N-terminal end compared to the parent GAA polypeptide. , especially at least 5, especially at least 6, especially at least 7, especially at least 8 consecutive amino acids. In another embodiment, the truncated GAA polypeptide has at most 75, in particular at most 70, in particular at most 60, especially at most 60, at its N-terminal end deleted compared to said parent GAA polypeptide. 55, in particular at most 50, in particular at most 47, in particular at most 46, in particular at most 45, in particular at most 44, in particular at most 43, consecutive amino acids. In other specific embodiments, said truncated GAA polypeptide has at most 47, in particular at most 46, in particular at most 45, especially at most 45, at its N-terminal end deleted compared to said parent GAA polypeptide. 44, especially up to 43 consecutive amino acids. In another specific embodiment, the truncated GAA polypeptide has from 1 to 75, in particular from 1 to 47, especially from 1 to 46, deleted at its N-terminal end compared to the parent GAA polypeptide. , especially 1 to 45, especially 1 to 44, especially 1 to 43 consecutive amino acids. In another embodiment, the truncated GAA polypeptide has 2 to 43, in particular 3 to 43, especially 4 to 43, deleted at its N-terminal end compared to the parent GAA polypeptide. In particular 5 to 43, especially 6 to 43, especially 7 to 43, especially 8 to 43 consecutive amino acids. In a more specific embodiment, the truncated GAA polypeptide has 6, 7, 8, 9, 10, 27, 28, 29, 30, 31, 40 deleted at its N-terminal end compared to the parent GAA polypeptide. , 41, 42, 43, 44, 45, 46 or 47 consecutive amino acids, especially 7, 8, 9, 28, 29, 30, 41, truncated at its N-terminal end compared with the parent GAA polypeptide. 42, 43 or 44, more particularly 8, 29, 42 or 43 consecutive amino acids. In other specific embodiments, the parent polypeptide is human GAA (hGAA), particularly hGAA having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 33, particularly SEQ ID NO: 1.

In specific embodiments, the truncated GAA polypeptides of the invention have the sequences set forth in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, and SEQ ID NO:35.

Furthermore, the truncated GAA polypeptide of the invention may also comprise a signal peptide fused to its N-terminal end, in particular a signal peptide selected from the group consisting of SEQ ID NO: 3 to 7, in particular the signal peptide of SEQ ID NO: 3.

In another aspect, the invention relates to a nucleic acid molecule encoding a truncated GAA polypeptide as described above, optionally fused via its N-terminal end to a signal peptide. In certain embodiments, the nucleic acid molecules have properties optimized to increase expression of the truncated GAA polypeptide and/or increase immune tolerance to the truncated GAA polypeptide in vivo, particularly in human subjects. sexual nucleotide sequence.

In another aspect, the invention relates to a nucleic acid construct comprising a nucleic acid construct operably linked to one or more regulatory sequences such as a promoter, an intron, a polyadenylation signal and/or an enhancer (e.g., a cis-regulatory module or CRM) of the nucleic acid molecule of the present invention. In a specific embodiment, the promoter is preferably selected from the group consisting of alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter and thyroxine binding globulin (TBG) promoter liver-specific promoter. In another specific embodiment, the promoter is a muscle-specific promoter such as the Spc5-12, MCK and desmin promoters. In another embodiment, the promoter is a ubiquitous promoter such as the CMV, CAG and PGK promoters. The nucleic acid construct may also optionally comprise an intron, in particular selected from the group consisting of human β-globin b2 (or HBB2) intron, FIX intron, chicken β-globin intron and SV40 intron. An intron, wherein the intron is optionally a modified intron such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19 or the modified FIX intron of SEQ ID NO: Modified chicken beta-globin intron of 21. In a specific embodiment of the nucleic acid construct of the invention, the nucleic acid construct preferably comprises in the following order: an enhancer; an intron; a promoter, in particular a liver-specific promoter; a nucleic acid encoding the GAA protein sequence; and a polyadenylation signal, said construct preferably comprising in the following order: an ApoE control region; an HBB2 intron, in particular a modified HBB2 intron; an hAAT promoter; encoding said truncated GAA polypeptide Nucleic acid sequence of; and bovine growth hormone polyadenylation signal. In a specific embodiment, the nucleic acid construct more specifically comprises the nucleotide sequence of any one of SEQ ID NOs: 22 to 26.

In another aspect, the invention relates to a vector comprising a nucleic acid molecule or nucleic acid construct disclosed herein. The vector of the present invention may in particular be a viral vector, preferably a retroviral vector such as a lentiviral vector, or an AAV vector. Preferably, the vector is a single-stranded or double-stranded self-complementary AAV vector, preferably a capsid having an AAV origin such as AAV1, AAV2, variant AAV2, AAV3, variant AAV3, AAV3B, variant AAV3B, AAV4, AAV5 , AAV6, variant AAV6, AAV7, AAV8, AAV9, AAV10 such as AAVcy10 and AAVrh10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8 and porcine AAV such as AAVpo4 and AAVpo6 capsids or AAV vectors with chimeric capsids . In a specific embodiment, the vector is an AAV vector having an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, particularly an AAV8, AAV9 or AAVrh74 capsid, more particularly an AAV8 capsid.

In another aspect, the invention provides a cell transformed with a nucleic acid molecule, nucleic acid construct or vector of the invention. More specifically, the cells are hepatocytes or myocytes.

In certain instances, the invention provides a pharmaceutical composition comprising a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention in a pharmaceutically acceptable carrier.

The invention also relates to a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention for use as a medicament.

The invention also provides a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention for use in a method of treating glycogen storage disease. In specific embodiments, the glycogen storage disease is GSDI, GSDII, GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or a lethal congenital glycogen storage disease of the heart. In a more specific embodiment, the glycogen storage disease is selected from GSDI, GSDII and GSDIII, more particularly from GSDII and GSDIII. In an even more specific embodiment, the glycogen storage disease is GSDII.

Description of drawings

Figure 1. Partial deletion of hGAA improves its secretion in vitro. Figure A. Using Lipofectamine ^TM , human hepatoma cells (Huh7) were treated with a control plasmid expressing green fluorescent protein (GFP) or expressing wild-type hGAA (hGAA) or hGAA sequence-optimized according to two different algorithms (hGAAco1, respectively). and co2) plasmid transfection. Different hGAA constructs contain wild-type or human α-1-antitrypsin signal peptide (sp2). Truncated hGAA is obtained by deleting 8 amino acids after the signal peptide (Δ8). At 48 hours after transfection, hGAA activity in the culture medium was measured by luciferase assay and GAA activity was assessed against a standard curve of reaction products specified in “Materials and Methods”. The bar graph shows the mean ± SE of secreted hGAA levels derived from three different experiments. Statistical analysis was performed by paired t-test and the obtained p-values are reported in histograms (*=p<0.05 as indicated). Panel B. Human hepatoma cells (Huh7) were transfected with a control plasmid expressing GFP or a plasmid expressing hGAAco1 with wild-type or chymotrypsinogen B1 signal peptide (sp7) using Lipofectamine. The hGAA protein has been truncated by removing 8 or 42 amino acids after the signal peptide (Δ8 and Δ42, respectively). 48 hours after transfection, hGAA activity in the culture medium was measured by the fluorogenic assay as indicated above. The bar graph shows the mean ± SE of secreted hGAA levels derived from three different experiments. Statistical analysis was performed by ANOVA (*=p<0.05 as indicated).

Figure 2. Partial deletion of hGAA enhances its secretion in the bloodstream in a mouse model of Pompe disease. Three-month-old GAA ^−/− mice (n = 4-5 mice/group) were injected intravenously with PBS or 2E12 vg/kg of AAV8 vector expressed under the transcriptional control of a liver-specific promoter. Sequence optimized hGAA (hGAAco1). The wild-type signal peptide of hGAA has been replaced with the chymotrypsinogen signal peptide (sp7), and the sequence of hGAA is used as the full-length native sequence or truncated by removing 8 or 42 amino acids after the signal peptide (Δ8, respectively and Δ42). One month after injection, the mice were bled, and hGAA activity in the serum was measured using a fluorogenic assay. Statistical analysis was performed by ANOVA (*=p<0.05 as indicated).

Figure 3. Signal peptide enhances secretion of hGAA. Human hepatoma cells (Huh7) were treated with a control plasmid (GFP), a plasmid expressing wild-type hGAA (termed sp1), or a sequence optimized for expression fused to signal peptide 6-8 (sp6-8) by Lipofectamine ^™ . Plasmid transfection of Δ8hGAA (hGAAco). At 48 hours after transfection, hGAA activity in the culture medium was measured by luciferase assay and GAA activity was assessed against a standard curve for 4-methylumbelliferone. The bar graph shows the mean ± SE of secreted hGAA levels derived from three different experiments. Statistical analysis was performed by ANOVA (*=p<0.05 vs. mock-transfected cells).

Figure 4. Truncated Δ8hGAA efficiently corrects glycogen accumulation in a mouse model of Pompe disease. Four-month-old wild-type (WT) and GAA ^−/− mice (n = 4-5 mice/group) were injected intravenously with PBS or 6E11 vg/kg of AAV8 vector, which is expressed in human α-1- Sequence-optimized Δ8hGAA (hGAAco) fused to signal peptides 1, 2, 7, and 8 (sp1, 2, 7, 8) was expressed under the transcriptional control of the antitrypsin promoter. Panel A. Bar graph showing hGAA activity measured in blood by fluorometry 3 months after vector injection. Statistical analysis was performed by ANOVA, and the p-values obtained relative to PBS-treated GAA-/- animals are reported in the bar graphs (*=p<0.05). Figure BD. Biochemical correction of glycogen content in the heart, diaphragm, and quadriceps muscles. Four-month-old GAA ^−/− mice were processed as described above. Three months after injection, mice were sacrificed and glycogen content was assessed. The bar graph shows the glycogen content measured in the heart (Panel B), diaphragm (Panel C) and quadriceps muscle (Panel D), expressed as glucose released after enzymatic digestion of glycogen. Statistical analysis was performed by ANOVA (*=p<0.05 vs. PBS-injected GAA-/- mice).

Figure 5. Highly secreted hGAA reduces humoral responses in a mouse model of Pompe disease. Four-month-old GAA-/- mice were injected intravenously with PBS or two different doses (5E11 or 2E12 vg/kg) of the AAV8 vector contained under the transcriptional control of the human α-1-antitrypsin promoter. The following is an optimized sequence encoding Δ8hGAA fused to signal peptide 1 (co), signal peptide 2 (sp2-Δ8-co), signal peptide 7 (sp7-Δ8-co), or signal peptide 8 (sp8-Δ8-co). One month after injection, sera were analyzed for the presence of anti-hGAA antibodies by ELISA. Quantification was performed using purified mouse IgG as standard. Statistical analysis was performed by ANOVA and Dunnett's post hoc test (*=p<0.01).

Figure 6. AAV8-hAAT-sp7-Δ8-hGAAco1 injection causes efficient secretion of hGAA in blood and uptake in muscle in NHP. Two cynomolgus monkeys (Macaca Fascicularis) were injected on day 0 with 2E12 vg/kg of AAV8-hAAT-sp7-Δ8-hGAAco1. Panel A Western blot of hGAA performed on sera obtained from two monkeys 12 days before and 30 days after vehicle administration. Band positions for molecular weight markers run parallel to the sample are indicated on the left. Panel B Three months after vector injection, monkeys were sacrificed and tissues were harvested for biochemical assessment of hGAA uptake. hGAA Western blotting was performed on tissue extracts obtained from biceps and diaphragm. Anti-tubulin antibody was used as a loading control. Band positions for molecular weight markers run parallel to the sample are indicated on the left.

Figure 7. Biochemical calibration of glycogen content in livers of GDE-/- animals injected with hGAA expression vector. Three-month-old wild-type (WT) or GDE-/- mice were treated with PBS or AAV8 expressing codon-optimized hGAA under the transcriptional control of the human α-1-antitrypsin promoter and fused to signal peptide 7. The vector (AAV8-hAAT-sp7--Δ8-hGAAco1) was injected intravenously at a dose of 1E11 or 1E12 vg/mouse. The bar graph shows the glycogen content measured in the liver, expressed as glucose released after enzymatic digestion of glycogen. Statistical analysis was performed by ANOVA (*=p<0.05 vs. PBS-injected GDE-/- mice, §=p<0.05 vs. PBS-injected WT animals).

Figure 8. GAA activity in the culture medium of cells transfected with plasmids encoding different GAA variants. GAA activity was measured in the culture medium of HuH7 cells at 24 hours (Panel A) and 48 hours (Panel B) after transfection with a plasmid containing a plasmid encoding native GAA(co) in combination with a native GAA sp1 signal peptide or encoding including Optimized sequence of engineered GAA(sp7-co) of native GAA combined with heterologous sp7 signal peptide. The effect of different deletions in the GAA coding sequence following the sp7 signal peptide was evaluated (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7- Δ62-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test. Hash symbols (#) in bars show statistically significant differences relative to co; tau symbols (τ) show statistically significant differences relative to sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7 -Statistically significant difference for Δ43-co. Data are means ± SD of two independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001, except when different signs are used.

Figure 9. Intracellular GAA activity of different GAA variants. GAA activity was measured in lysates of HuH7 cells 48 hours after transfection with plasmids containing plasmids encoding native GAA(co) in combination with native GAA sp1 signal peptide or encoding native GAA in combination with heterologous sp7 signal peptide. Optimized sequence of engineered GAA(sp7-co). The effect of different deletions in the GAA coding sequence following the signal peptide was evaluated (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7 -Δ62-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test. Tau symbols (τ) show statistically significant differences relative to sp7-co, sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co. Data are means ± SD of two independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001, except when different signs are used.

Figure 10. Improved GAA activity in cell culture medium using Δ8 deletion in combination with sp6 or sp8 signal peptide. GAA activity was measured in the culture medium (Panel A) and lysate (Panel B) of HuH7 cells 48 hours after transfection with a plasmid containing a plasmid encoding native GAA (co) in combination with native GAA sp1 signal peptide or encoding Optimized sequences of engineered GAA (sp6-co or sp8-co) including native GAA combined with heterologous sp6 or sp8 signal peptide. The effect of deletion of 8 amino acids in the GAA coding sequence following the signal peptide was evaluated (sp6-Δ8-co, sp8-Δ8-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test. Asterisks in bars show statistically significant differences relative to co. Data are means ± SD of two independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001, except when different signs are used.

Detailed description of the invention

The present invention relates to a truncated GAA polypeptide, a nucleic acid molecule encoding such a truncated GAA polypeptide, a nucleic acid construct comprising the nucleic acid, a vector comprising the nucleic acid construct, a nucleic acid molecule or construct comprising the nucleic acid molecule or cells of the vector, and pharmaceutical compositions comprising the polypeptides, nucleic acid molecules, nucleic acid constructs, vectors or cells of the invention. The inventors have surprisingly shown that the truncated form of GAA of the present invention greatly increases GAA secretion while simultaneously reducing its immunogenicity.

Lysosomal acid alpha-glucosidase or "GAA" (E.C.3.2.1.20) (1,4-alpha-D-glucan glucohydrolase) is an exo-1,4-alpha-D-glucose Glycodase, which hydrolyzes both the α-1,4 and α-1,6 linkages of oligosaccharides to release glucose. A deficiency in GAA causes glycogen storage disease type II (GSDII), also known as Pompe disease (although this term previously referred to the infantile-onset form of the disease). It catalyzes the complete degradation of glycogen and slows it down at the branch point. The 28 kb human acid alpha-glucosidase gene on chromosome 17 encodes a 3.6 kb mRNA that produces a 952 amino acid polypeptide (Hoefsloot et al., (1988) EMBO J. 7:1697; Martiniuk et al., (1990) DNA and Cell Biology 9:85). The enzyme undergoes N-linked glycosylation in the endoplasmic reticulum concurrent with translation. It is synthesized as a 110-kDa precursor form, which undergoes extensive glycosylation modification, phosphorylation, and undergoes proteolytic processing through an approximately 90-kDa endosomal intermediate to mature into the final lysosomal 76 and 67 kDa Form (Hoefsloot, (1988) EMBO J. 7:1697; Hoefsloot et al., (1990) Biochem. J. 272:485; Wisselaar et al., (1993) J. Biol. Chem. 268:2223; Hermans et al., (1993) Biochem. J. 289:681).

In patients with GSD II, a deficiency of acid alpha-glucosidase causes glycogen to accumulate in lysosomes, disrupting cellular function (Hirschhorn, R. and Reuser, A.J., (2001), in "Genetic Diseases" "The Metabolic and Molecular Basis for Inherited Disease", edited by Scriver, C.R. et al., pp. 3389-3419 (McGraw-Hill, New York). In the most common infantile form, patients exhibit progressive muscle degeneration and cardiomyopathy and die before the age of 2 years. In adolescent and adult-onset forms, there is severe debilitation.

Additionally, patients with other GSDs may also benefit from administration of optimized forms of GAA. For example, it has been shown (Sun et al., (2013) Mol Genet Metab 108(2):145; WO2010/005565) that administration of GAA in primary myoblasts from patients with glycogen storage disease type III (GSD III) Moderately lower glycogen.

In particular, in the context of the present invention, a "precursor form of GAA" is a form of a GAA polypeptide that contains its native signal peptide. For example, the sequence of SEQ ID NO: 2 is the precursor form of human GAA (hGAA). In SEQ ID NO: 2, amino acid residues 1-27 correspond to the signal peptide of the hGAA polypeptide. The sequence of this hGAA signal peptide is also shown in SEQ ID NO:4.

In the context of the present invention, the truncated GAA polypeptides of the invention are derived from the parent GAA polypeptide. According to the present invention, a "parent GAA polypeptide" may be a functional precursor GAA sequence as defined above, but without its signal peptide. For example, with reference to a typical wild-type human GAA polypeptide, the complete wild-type GAA polypeptide (i.e., the precursor form of GAA) is shown in SEQ ID NO: 2 or SEQ ID NO: 30 and has a signal peptide (corresponding to SEQ ID NO: 2 or amino acids 1-27 of SEQ ID NO: 30), whereas the parent GAA polypeptides that serve as the basis for the truncated GAA forms of these wild-type human GAA polypeptides are shown in SEQ ID NO: 1 and SEQ ID NO: 33, respectively, And there is no signal peptide. In this example, the latter corresponding to amino acids 28-952 of SEQ ID NO:2 and amino acids 28-952 of SEQ ID NO:30 are referred to as the parent GAA polypeptide.

According to the present invention, the truncated GAA polypeptide of the invention is a functional GAA polypeptide, that is, it has the functions of a wild-type GAA polypeptide. As defined above, the function of wild-type GAA is to hydrolyze both oligosaccharides and polysaccharides, more particularly both the alpha-1,4 and alpha-1,6 chains of glycogen, to release glucose. The functional GAA protein encoded by the nucleic acid of the present invention can have at least 50%, 60%, 70%, 80%, 90%, 95% of the wild-type GAA polypeptide of SEQ ID NO: 1 or SEQ ID NO: 33. , 99% or at least 100% glycogen hydrolytic activity. The activity of the GAA protein encoded by the nucleic acid of the invention may even be more than 100% of the activity of the wild-type GAA protein of SEQ ID NO: 1 or SEQ ID NO: 33, for example more than 110%, 120%, 130%, 140% or Even more than 150%.

The amino acid sequence of the parent GAA polypeptide or its coding sequence can be derived from any source, including avian and mammalian species. As used herein, the term "birds" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. As used herein, the term "mammal" includes, but is not limited to, humans, apes and other non-human primates, bovines, sheep, goats, horses, felines, canines, lagomorphs, and the like. . In an embodiment of the invention, the parent GAA polypeptide is a human, mouse or quail, particularly a human GAA polypeptide.

Furthermore, the parent GAA polypeptide may be a functional variant of the GAA polypeptide that contains one or more amino acid modifications such as amino acid insertions, deletions and/or substitutions compared to the GAA polypeptide. For example, the parent polypeptide may be a functional derivative of a human GAA polypeptide such as SEQ ID NO: 1 or SEQ ID NO: 33, especially a polypeptide of SEQ ID NO: 1, that shares at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity. For example, in addition to the truncations defined above, functional variants of a GAA polypeptide are identical to said parent polypeptide, for example the parent set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 GAA polypeptides may have between 0 and 50, between 0 and 30, between 0 and 20, between 0 and 15, between 0 and 10, or between 0 and 5 amino acid changes. In particular, the parent GAA polypeptide may consist of a human GAA polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

The term "identity" and its deviations when referring to polypeptides means that when a position in the two polypeptide sequences being compared is occupied by the same amino acid (for example if a position in each of the two polypeptides is occupied by a leucine occupies), then the polypeptide has identity at that position. The percent identity between two polypeptides is a function of the number of matching positions common to the two sequences divided by the number of positions compared X 100. For example, if 6 out of 10 positions in two polypeptides match, the two sequences are 60% identical. Typically, comparisons are made when two sequences are aligned to give maximum identity. A variety of different bioinformatics tools known to those skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

The parent GAA polypeptide may also be a GAA variant, such as GAA II as described by Kunita et al., (1997) Biochemica et Biophysica Acta 1362:269; by Hirschhorn, R. and Reuser, A.J. (2001) Metabolism of Genetic Diseases GAA described in The Metabolic and Molecular Basis for Inherited Disease (Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., editors), pp. 3389-3419, McGraw-Hill, New York Polymorphisms and SNPs, see pages 3403-3405. Any variant GAA polypeptide known in the art can be used as a basis for defining the parent GAA polypeptide. Exemplary variant GAA polypeptides include SEQ ID NO: 2 (NCBI reference sequence NP_000143.2); SEQ ID NO: 29 (GenBank AAA52506.1); SEQ ID NO: 30 (GenBank CAA68763.1); SEQ ID NO: 31 (GenBank: EAW89583.1) and SEQ ID NO: 32 (GenBankABI53718.1). Other useful variants are included in Hoefsloot et al., (1988) EMBO J. 7:1697 and Van Hove et al., (1996) Proc. Natl. Acad. Sci. USA 93:65 (human) and GenBank accession number NM_008064 (mouse ) as described in. Other variant GAA polypeptides include those described in WO2012/145644, WO00/34451 and US6,858,425. In specific embodiments, the parent GAA polypeptide is derived from the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:30.

Truncated forms of GAA of the present invention are N-terminally truncated forms of a parent GAA polypeptide in which at least one amino acid is deleted from the N-terminus of the parent GAA polypeptide.

"Truncated form" means a GAA polypeptide that contains one or several consecutive amino acids deleted from the N-terminal portion of the parent GAA polypeptide. For example, the GAA moiety may be truncated from its N-terminal end by 1 to 75 contiguous amino acids or by more than 75 contiguous amino acids compared to the parent GAA protein. Specifically, the truncated GAA polypeptide can be truncated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 from its N-terminal end compared with the parent GAA protein. ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36 ,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 consecutive amino acids (especially in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ A truncated form of the parent hGAA protein shown in ID NO: 1). Using alternative nomenclature, the GAA polypeptide resulting from the truncation of 1 amino acid in the parent GAA polypeptide is referred to as the Δ1 GAA truncated form, and the GAA polypeptide resulting from the truncation of 2 consecutive amino acids from the N-terminus is is called the Δ2GAA truncated form, the GAA polypeptide resulting from the truncation of 3 consecutive amino acids in the parent GAA polypeptide is called the Δ3GAA truncated form, and so on. In specific embodiments, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18 , Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43 , Δ44, Δ45, Δ46, Δ47, Δ48, Δ49, Δ50, Δ51, Δ52, Δ53, Δ54, Δ55, Δ56, Δ57, Δ58, Δ59, Δ60, Δ61, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68 , Δ69, Δ70, Δ71, Δ72, Δ73, Δ74 or Δ75GAA truncated forms (especially truncated forms of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 form).

In another specific embodiment, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17 , Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 , Δ43, Δ44, Δ45, Δ46 or Δ47 GA truncated form (especially the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17 , Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 , Δ43, Δ44, Δ45 or Δ46 GA truncated form (especially the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17 , Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 , Δ43, Δ44 or Δ45 GA truncated form (especially the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, A Δ43 or Δ44 GA truncated form (especially a truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, or Δ43GAA truncated form (in particular the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA Short forms (especially truncated forms of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA Short forms (especially truncated forms of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20,Δ21,Δ22,Δ23,Δ24,Δ25,Δ26,Δ27,Δ28,Δ29,Δ30,Δ31,Δ32,Δ33,Δ34,Δ35,Δ36,Δ37,Δ38,Δ39,Δ40,Δ41,Δ42 or Δ43GAA truncated form (In particular the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form (especially is a truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated forms (especially in SEQ ID NO: 1 or SEQ ID NO: 33, in particular a truncated form of the parent hGAA protein shown in SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated forms (especially in SEQ ID NO: 1 or SEQ ID NO: 33, in particular a truncated form of the parent hGAA protein shown in SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated forms (especially in SEQ ID NO: 1 or SEQ ID NO:33, in particular a truncated form of the parent hGAA protein shown in SEQ ID NO:1).

In other specific embodiments, the truncated GAA polypeptides of the invention are Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated forms (especially in SEQ ID NO: 1 or SEQ ID NO:33, in particular a truncated form of the parental hGAA protein shown in SEQ ID NO:1).

In other specific embodiments, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9 or Δ10 truncated forms, in particular Δ7, Δ8 or Δ9 of GAA (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1) A truncated form, more particularly a Δ8 truncated form of GAA (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptide of the invention is Δ27, Δ28, Δ29, Δ30 or Δ31 truncated forms, in particular Δ28, Δ29 or Δ30 of GAA (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1) A truncated form, more particularly a Δ29 truncated form of GAA (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptide of the invention is the Δ40 of GAA (especially the hGAA protein set forth in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1) , Δ41, Δ42, Δ43 or Δ44 truncated forms, especially Δ41, Δ42 or truncated forms of GAA (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1) A Δ43 truncated form, more particularly a Δ42 truncated form of GAA (especially the hGAA protein set forth in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1).

In other specific embodiments, the truncated GAA polypeptide of the invention is a Δ41, Δ42, Δ43, Δ44 or Δ45 truncated form of GAA (especially the hGAA protein set forth in SEQ ID NO: 1), especially GAA Δ42, Δ43 or Δ44 truncated form of the hGAA protein (especially the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1), more particularly GAA (especially the hGAA protein shown in SEQ ID NO: 1 : Δ43 truncated form of hGAA protein shown in 1).

In another embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ27, Δ28, Δ29, Δ30, Δ31, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46 or Δ47 truncated forms.

In another embodiment, the truncated GAA polypeptide of the invention is Δ7, Δ8, Δ9, Δ28, Δ29, Δ30, Δ41, Δ42, Δ43 or Δ44 truncated forms.

In another embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ40, Δ41, Δ42, Δ43 or Δ44 truncated forms.

In another embodiment, the truncated GAA polypeptide of the invention is Δ8, Δ29, Δ42, Δ43 or Δ47 truncated forms.

In another embodiment, the truncated GAA polypeptide of the invention is Δ8, Δ29, Δ42 or Δ43 truncated forms.

In another embodiment, the truncated GAA polypeptide of the invention is Δ8 or Δ42 truncated form.

In specific embodiments of the invention, the truncated GAA polypeptides of the invention are truncated forms of functional human GAA polypeptides. In other specific embodiments, the parent hGAA polypeptide is the hGAA polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO: 33, particularly SEQ ID NO: 1. In a variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly represented in SEQ ID NO: 1 Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 of the hGAA polypeptide or functional variant thereof , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45 , Δ46, Δ47, Δ48, Δ49, Δ50, Δ51, Δ52, Δ53, Δ54, Δ55, Δ56, Δ57, Δ58, Δ59, Δ60, Δ61, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68, Δ69, Δ70 , Δ71, Δ72, Δ73, Δ74 or Δ75GAA truncated form, the functional variant comprising an amino acid substitution in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and At least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 sex.

In a variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly represented in SEQ ID NO: 1 Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 of the hGAA polypeptide or functional variant thereof , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45 , Δ46 or Δ47GAA truncated forms, the functional variants comprise amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In a variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly represented in SEQ ID NO: 1 Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 of the hGAA polypeptide or functional variant thereof , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45 Or a truncated form of Δ46GAA, the functional variant comprising an amino acid substitution in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and being identical to SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In a variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly represented in SEQ ID NO: 1 Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 of the hGAA polypeptide or functional variant thereof , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44 or Δ45GAA Truncated forms, said functional variants comprise amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO :33, especially SEQ ID NO:1 having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, or Δ44GAA truncated form, the functional variant comprising amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO:33, particularly SEQ ID NO:1, has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA Short form, said functional variant comprises amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO: 33. In particular, SEQ ID NO: 1 has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19,Δ20,Δ21,Δ22,Δ23,Δ24,Δ25,Δ26,Δ27,Δ28,Δ29,Δ30,Δ31,Δ32,Δ33,Δ34,Δ35,Δ36,Δ37,Δ38,Δ39,Δ40,Δ41 or Δ42GAA truncated form , the functional variant contains amino acid substitutions in SEQ ID NO: 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO: 33, In particular, SEQ ID NO: 1 has an identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, so Said functional variants comprise amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 is at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the function Sexual variants comprise amino acid substitutions in the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the functional changes The entity contains amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the functional variant is in The sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has the same amino acid substitution as SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 At least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the functional variant is in SEQ ID NO. : 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, contains amino acid substitutions, and has at least 75 or 80 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the functional variant is in SEQ ID NO: 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, containing amino acid substitutions, and having at least 75 or 80 % differences with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42GAA truncated form, the functional variant is in SEQ ID NO: 1 or The sequence shown in SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and is at least 75, 80, 85, 90 with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20,Δ21,Δ22,Δ23,Δ24,Δ25,Δ26,Δ27,Δ28,Δ29,Δ30,Δ31,Δ32,Δ33,Δ34,Δ35,Δ36,Δ37,Δ38,Δ39,Δ40,Δ41,Δ42 or Δ43GAA truncated form , the functional variant contains amino acid substitutions in SEQ ID NO: 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO: 33, In particular, SEQ ID NO: 1 has an identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, so Said functional variants comprise amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 is at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, the function Sexual variants comprise amino acid substitutions in the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and are identical to SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, the functional changes The entity contains amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, and is identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, the functional variant is in The sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has the same amino acid substitution as SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 At least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, the functional variant is in SEQ ID NO. : 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, contains amino acid substitutions, and has at least 75 or 80 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly in SEQ ID NO: Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or Δ43GAA truncated form, the functional variant is in SEQ ID NO: 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1, containing amino acid substitutions, and having at least 75 or 80 % differences with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ6, Δ7, Δ8, Δ9 or Δ10, especially Δ7, Δ8 or Δ9, more particularly Δ8 truncated forms of hGAA polypeptides or functional variants thereof, said functional variants in SEQ ID NO: 1 or The sequence shown in SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has at least 80, 85 or 90 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ27, Δ28, Δ29, Δ30 or Δ31, especially Δ28, Δ29 or Δ30, more particularly Δ29 truncated forms of the hGAA polypeptide or functional variants thereof, said functional variants in SEQ ID NO: 1 or The sequence shown in SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has at least 80, 85 or 90 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ40, Δ41, Δ42, Δ43 or Δ44, especially Δ41, Δ42 or Δ43, more particularly Δ42 truncated forms of the hGAA polypeptide or functional variants thereof, said functional variants in SEQ ID NO: 1 or The sequence shown in SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has at least 80, 85 or 90 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ41, Δ42, Δ43, Δ44 or Δ45, especially Δ42, Δ43 or Δ44, more particularly Δ43 truncated forms of the hGAA polypeptide or functional variants thereof, said functional variants in SEQ ID NO: 1 or The sequence shown in SEQ ID NO: 33, especially SEQ ID NO: 1, contains amino acid substitutions and has at least 80, 85 or 90 0% similarity with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 , 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ6, Δ7, Δ8, Δ9, Δ10, Δ27, Δ28, Δ29, Δ30, Δ31, Δ40, Δ41, Δ42, Δ43, Δ44 or Δ45 of the hGAA polypeptide or functional variant thereof, especially Δ7, Δ8, Δ9 , Δ28, Δ29, Δ30, Δ41, Δ42, Δ43 or Δ44, especially Δ8, Δ29, Δ42 or Δ43 truncated forms, the functional variants are in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ The sequence shown in ID NO: 1 contains amino acid substitutions and has at least 80, 85, 90, 95, 96, 97, 98 or more similarities with SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 99% identical.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ6, Δ7, Δ8, Δ9, Δ10, Δ40, Δ41, Δ42, Δ43 or Δ44, especially Δ8 or Δ42 truncated forms of the hGAA polypeptide or functional variants thereof, which functional variants are in SEQ ID NO. : 1 or SEQ ID NO: 33, especially the sequence shown in SEQ ID NO: 1 contains amino acid substitutions, and has at least 80, 85, 90, 95, 96, 97, 98 or 99% identity.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ8, Δ29, Δ42, Δ43 or Δ47 truncated form of the hGAA polypeptide or a functional variant thereof, which functional variant is represented by SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 The sequence shown contains amino acid substitutions and is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 Δ8, Δ29, Δ42 or Δ43 truncated form of the hGAA polypeptide or a functional variant thereof, the functional variant is shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 The sequence contains amino acid substitutions and is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 33, particularly SEQ ID NO: 1.

In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly represented in SEQ ID NO: 1 or SEQ ID NO: 33, particularly in SEQ ID NO: 1 A Δ8 or Δ42 truncated form of the hGAA polypeptide or a functional variant thereof comprising amino acids in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1 substitution, and is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 33, especially SEQ ID NO: 1.

In specific embodiments, the truncated hGAA polypeptide of the invention has the sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36 or an amino acid sequence consisting of a functional variant thereof, which functional variant is identical to that shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36 The sequence comparison contains 1 to 5, in particular 1 to 4, in particular 1 to 3, more in particular 1 to 2, in particular 1 amino acid substitutions. In another specific embodiment, the truncated hGAA polypeptide of the invention has the sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35 or the functionality thereof Amino acid sequences consisting of variants, said functional variants comprising 1 to 5 amino acids compared to the sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35 replace. In a specific embodiment, the truncated hGAA polypeptide of the invention has an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28, or a functional variant thereof, which functional variant is identical to The sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28 contains 1 to 5, especially 1 to 4, especially 1 to 3, more particularly 1 to 2, especially 1 Amino acid substitutions.

The truncated GAA polypeptides of the invention may also comprise a signal peptide, such as the native signal peptide of GAA or an alternative signal peptide derived from another secreted protein. Non-limiting examples of these signal peptides include those set forth in SEQ ID NOs: 3 to 7. The inventors have surprisingly shown that fusing a truncated GAA polypeptide of the invention to an alternative signal peptide enhances its secretion even further. Accordingly, the present invention provides a chimeric GAA polypeptide comprising a signal moiety and a truncated GAA polypeptide moiety, the truncated GAA polypeptide moiety being a truncated GAA polypeptide as defined above. In a specific embodiment, the signal peptide is a native signal peptide of GAA, such as the signal peptide of hGAA set forth in SEQ ID NO:4. In another embodiment, the signal peptide is an exogenous (or alternative) signal peptide derived from a protein other than GAA. In a specific embodiment, the alternative signal peptide is selected from the group consisting of SEQ ID NOs: 3, 5, 6 and 7 or functional derivatives thereof as defined below.

The inventors have shown that an exogenous signal peptide fused to the remainder of the GAA protein increases secretion of the resulting chimeric GAA polypeptide compared to the corresponding GAA polypeptide containing its native signal peptide. In addition, the truncated GAA polypeptide moiety also has improved performance compared to a chimeric GAA polypeptide comprising the same signal peptide fused to the parent GAA polypeptide. secretion of both polypeptides).

Specific signal peptides that may work in the present invention include amino acids 1-20 from chymotrypsinogen B2 (SEQ ID NO: 3), the signal peptide from human alpha-1-antitrypsin (SEQ ID NO: 5 ), amino acids 1-25 from iduronate-2-sulfatase (SEQ ID NO: 6) and amino acids 1-23 from protease C1 inhibitor (SEQ ID NO: 7). The signal peptides of SEQ ID NO:3 and SEQ ID NO:5 to SEQ ID NO:7 allow for higher secretion of the chimeric GAA protein both in vitro and in vivo when compared to GAA containing the native signal peptide. In a specific embodiment, the signal peptide has the sequence set forth in SEQ ID NO: 3 to 7, or is a functional derivative thereof, that is, compared to the sequence set forth in SEQ ID NO: 3 to 7, it contains 1 to 5, in particular 1 to 5, in particular 1 to 3, more in particular 1 to 2, in particular 1, amino acid deletions, insertions or substitutions in the sequence, provided that the resulting sequence corresponds to a functional signal peptide , that is, the signal peptide that allows GAA protein to be secreted. In a specific embodiment, the signal peptide component sequence consists of a sequence selected from SEQ ID NO: 3 to 7.

In a specific embodiment, the GAA polypeptide of the invention is selected from:

- a combination of SEQ ID NO: 3 with a Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- a combination of SEQ ID NO: 4 with a Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- a combination of SEQ ID NO: 5 with a Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- a combination of SEQ ID NO: 6 with a Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- a combination of SEQ ID NO: 7 with a Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- a combination of SEQ ID NO: 3 with a Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- a combination of SEQ ID NO: 4 with a Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- a combination of SEQ ID NO: 5 with a Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- a combination of SEQ ID NO: 6 with a Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- a combination of SEQ ID NO: 7 with a Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- a combination of SEQ ID NO: 3 with a Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- a combination of SEQ ID NO: 4 with a Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- a combination of SEQ ID NO: 5 with a Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- a combination of SEQ ID NO: 6 with a Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- a combination of SEQ ID NO: 7 with a Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- a combination of SEQ ID NO: 3 with a Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- a combination of SEQ ID NO: 4 with a Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- a combination of SEQ ID NO: 5 with a Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- a combination of SEQ ID NO: 6 with a Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35; and

- a combination of SEQ ID NO: 7 with a Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- a combination of SEQ ID NO: 3 with a Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- a combination of SEQ ID NO: 4 with a Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- a combination of SEQ ID NO: 5 with a Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- a combination of SEQ ID NO: 6 with a Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36; and

- a combination of SEQ ID NO: 7 with a Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

Or a functional derivative thereof, which has at least 90% identity with the resulting sequence combination, especially at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Identity. In these embodiments, as mentioned above, the signal peptide component may comprise from 1 to 5, in particular from 1 to 4, especially from the sequence shown in SEQ ID NO: 3 to 7. Sequences in which 1 to 3, more particularly 1 to 2, in particular 1 amino acid are deleted, inserted or substituted, allow the resulting chimeric truncated GAA protein as long as the resulting sequence corresponds to a functional signal peptide The secreted signal peptide is sufficient.

The relative proportions of newly synthesized GAA secreted from cells can be routinely determined by methods known in the art and described in the Examples. Secreted proteins can be detected by direct measurement of the protein itself in cell culture medium, serum, milk, etc. (eg by Western blotting) or by protein activity assays (eg enzymatic assays).

It will also be understood by those skilled in the art that the truncated GAA polypeptide or chimeric GAA polypeptide may contain additional amino acids, for example as a result of manipulations of nucleic acid constructs such as the addition of restriction sites, so long as these additional amino acids do not It is sufficient that the signal peptide or GAA polypeptide does not have a function. The additional amino acids may be cleaved or may be retained by the mature polypeptide so long as the retention does not result in a non-functional polypeptide.

In another aspect, the invention relates to a nucleic acid molecule encoding a truncated GAA polypeptide of the invention or a chimeric GAA polypeptide of the invention.

The sequence of the nucleic acid molecule of the invention encoding a truncated GAA is optimized for expression of the GAA polypeptide in vivo. Sequence optimization can include numerous changes to the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs) and reducing the number of splice donor and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. It is also known that the genetic code in different organisms often favors the use of one of several codons encoding the same amino acid over the others. With codon optimization, changes are introduced into a nucleotide sequence that take advantage of the codon preferences present in a given cellular context such that the resulting codon-optimized nucleotide sequence compares to a sequence that has not been codon-optimized is more likely to be expressed at relatively high levels in this given cellular context. In a preferred embodiment of the invention, this sequence-optimized nucleotide sequence encoding a truncated GAA is codon-optimized to match the non-codon-optimized nucleotide sequence encoding the same truncated GAA protein. Compared to improving its expression in human cells, for example by exploiting human-specific codon usage preferences.

In a specific embodiment, the optimized GAA coding sequence is codon-optimized and/or has increased GC content compared to nucleotides 82-2859 of the wild-type hGAA coding sequence of SEQ ID NO: 8. /or have a reduced number of alternative open reading frames and/or have a reduced number of splice donor and/or splice acceptor sites. For example, the nucleic acid sequence of the invention causes an increase in the GC content of the GAA sequence by at least 2, 3, 4, 5 or 10% compared to the sequence of the wild-type GAA sequence. In a specific embodiment, the nucleic acid sequence of the invention causes an increase in the GC content of said GAA sequence by 2, 3, 4 or more specifically 5% or 10% (especially 5%) compared to the sequence of the wild-type GAA nucleotide sequence. %). In a specific embodiment, the nucleic acid sequence of the invention encoding a functional GAA polypeptide is "substantially identical", i.e., has about 70% identity, to nucleotides 82-2859 of the sequence set forth in SEQ ID NO: 8. More preferably about 80% identity, even more preferably about 90% identity, even more preferably about 95% identity, even more preferably about 97%, 98% or even 99% identity. As mentioned above, in addition to GC content and/or ARF number, sequence optimization may also include a reduction in the number of CpG islands and/or a reduction in the number of splice donor and acceptor sites in the sequence. Of course, as is well known to those skilled in the art, sequence optimization is a balance between all these parameters, meaning that a sequence can be considered optimized if at least one of the above parameters improves while one or more other parameters do not. , as long as the optimized sequence results in an improvement of the transferred gene, such as an increase in expression and/or a reduction in the immune response to the transferred gene in vivo.

Furthermore, the adaptability of a nucleotide sequence encoding a functional GAA to the codon usage of human cells can be expressed as a codon adaptation index (CAI). The codon adaptation index is defined herein as a measure of the relative adaptability of a gene's codon usage to the codon usage of highly expressed human genes. The relative fitness (w) of each codon is the ratio of the usage of each codon to the usage of the most abundant codon for the same amino acid. The CAI is defined as the geometric mean of these relative fitness values. Exclude non-synonymous codons and stop codons (depending on the genetic code). CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15:1281-1295; see also: Kim et al., Gene. 1997, 199: 293-301; zurMegede et al., Journal of Virology, 2000, 74: 2628-2635). Preferably, the GAA-encoding nucleic acid molecule has a CAI of at least 0.75 (especially 0.77), 0.8, 0.85, 0.90, 0.92 or 0.94.

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single- or double-stranded form, in particular DNA encoding the GAA protein of the invention.

The inventors have discovered that the truncated GAA polypeptide described above, when expressed from the nucleic acid molecule encoding it, surprisingly causes high levels of functional GAA protein both in vitro and in vivo compared to wild-type GAA cDNA. Express. Furthermore, as also shown by the present inventors, truncated GAA proteins produced from liver and muscle cells expressing the nucleic acid molecules of the invention do not induce an immune response. This means that the nucleic acid molecules can be used to produce high levels of GAA polypeptides and provide therapeutic benefits, such as avoiding recourse to immunosuppressive treatments, allowing low doses of immunosuppressive treatments, and allowing repeated administration of the nucleic acids of the invention to subjects in need. molecular. Therefore, the truncated GAA polypeptides of the invention and the nucleic acid molecules of the invention are of particular interest in situations where a lack of GAA expression and/or activity or where high levels of GAA expression may ameliorate diseases, such as glycogen storage diseases. Specifically, the glycogen storage disease may be GSDI (von Gierke's disease), GSDII (Pompe's disease), GSDIII (Cori disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or fatal congenital glycogen storage diseases of the heart. More specifically, the glycogen storage disease is selected from GSDI, GSDII and GSDIII, even more particularly from GSDII and GSDIII. In an even more specific embodiment, the glycogen storage disease is GSDII. In particular, the nucleic acid molecules of the present invention can be used in gene therapy to treat GAA-deficient disorders or other disorders related to the accumulation of glycogen, such as GSDI (von Gierke's disease), GSDII (Pompe's disease) , GSDIII (Corey's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage diseases of the heart, more particularly GSDI, GSDII or GSDIII, even more particularly GSDII and GSDIII. In an even more specific embodiment, the nucleic acid molecules of the invention may be used in gene therapy to treat GSDII.

In another embodiment of the invention, the portion of the nucleic acid molecule of the invention encoding said truncated GAA polypeptide moiety is identical to the portion encoding SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 Corresponding portions of the nucleotide sequence have an identity of at least 75% (eg 77,7%) or at least 80% or at least 82% (eg 83.1%) to the sequence of a wild-type hGAA polypeptide without a signal peptide.

The truncated GAA component of the nucleic acid molecule of the invention preferably has at least 85%, more preferably at least 90%, even more preferably, the nucleotide sequence of SEQ ID NO: 10 or 11 as a sequence-optimized sequence. At least 92% identity, in particular at least 95% identity, for example at least 98, 99 or 100% identity.

The term "identity" and its deviations refer to the sequence identity between two nucleic acid molecules. When a position in two compared sequences is occupied by the same base (eg if a position in each of two DNA molecules is occupied by adenine), the molecules are identical at that position. The percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared X 100. For example, if 6 out of 10 positions in two sequences match, the two sequences are 60% identical. Typically, comparisons are made when two sequences are aligned to give maximum identity. A variety of different bioinformatics tools known to those skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

Furthermore, the nucleic acid molecule of the invention encodes a functional GAA protein, ie it encodes a human GAA protein which, when expressed, has the functionality of a wild-type GAA protein. As defined above, the function of wild-type GAA is to hydrolyze both oligosaccharides and polysaccharides, more particularly the alpha-1,4 and alpha-1,6 linkages of glycogen, to release glucose. The functional GAA protein encoded by the nucleic acid of the present invention may have at least 50%, 60%, 70%, 80%, 90%, 95% compared to the wild-type GAA protein of SEQ ID NO: 1, 2, 30 or 33. , 99% or at least 100% glycogen hydrolytic activity. The activity of the GAA protein encoded by the nucleic acid of the present invention may even be more than 100% of the activity of the wild-type GAA protein of SEQ ID NO: 1, 2, 30 or 33, such as more than 110%, 120%, 130%, 140% or Even more than 150%.

A skilled person can easily determine whether the nucleic acid of the invention expresses a functional GAA protein. Suitable methods will be apparent to those skilled in the art. For example, one suitable in vitro method involves inserting the nucleic acid into a vector such as a plasmid or viral vector, transfecting or transducing a host cell such as 293T or HeLa cells or other cells such as Huh7 with the vector, and assaying for GAA activity. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into a mouse model of Pompe disease or another glycogen storage disease and determining functional GAA and GAA in the mouse plasma. The presence of GAA in the organization. Suitable methods are described in more detail in the experimental section below.

In a specific embodiment, the nucleic acid molecule of the invention comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 27; in SEQ ID The sequence shown in NO: 48 or SEQ ID NO: 49, which encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 28; the sequence shown in SEQ ID NO: 50 or SEQ ID NO: 51, which encodes A polypeptide having the amino acid sequence set forth in SEQ ID NO: 35; or a sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53 encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 36 . In a further embodiment, the nucleic acid molecule of the invention comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 27; in SEQ ID NO : 48 or the sequence set forth in SEQ ID NO: 49, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 28; or the sequence set forth in SEQ ID NO: 50 or SEQ ID NO: 51, It encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:35. In a specific embodiment, the nucleic acid molecule of the invention comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 27.

The invention also relates to nucleic acid constructs comprising the nucleic acid molecules of the invention. The nucleic acid construct may correspond to an expression cassette comprising other sequences operably linked to one or more expression control sequences and/or other sequences that increase expression of the transferred gene and/or sequences that enhance secretion of the encoded protein and/or Nucleic acid sequences of the invention enhance the uptake of the encoded protein. When used herein, the term "operably linked" means that polynucleotide elements are linked in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or another transcriptional control sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. These expression control sequences are known in the art, such as promoters, enhancers (eg, cis-regulatory modules (CRM)), introns, polyA signals, and the like.

In particular, the expression cassette may comprise a promoter. The promoter may be a ubiquitous or tissue-specific promoter, in particular a promoter capable of promoting expression in cells or tissues requiring GAA expression, such as in cells or tissues requiring GAA expression in a GAA-deficient patient. In specific embodiments, the promoter is a liver-specific promoter such as alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), transthyretin promoter, albumin promoter, thyroxine Binding globulin (TBG) promoter, LSP promoter (containing thyroxine-binding globulin promoter sequence, two copies of α1-microglobulin/bikunin enhancer sequence and leader sequence -34. Ill, C.R. et al., (1997), Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A, Blood Coag. Fibrinol. 8:S23–S30), etc. Other useful liver-specific promoters are known in the art, for example in the liver-specific gene promoter database compiled by Cold Spring Harbor Laboratory (http://rulai.cshl.edu/LSPD/ ). In the context of the present invention, a preferred promoter is the hAAT promoter. In another embodiment, the promoter is one that directs expression in a tissue or cell of interest (eg, in muscle cells) and in hepatocytes. For example, to a certain extent, promoters specific to muscle cells, such as desmin, Spc5-12 and MCK promoters, may have a certain expression leakage in hepatocytes, which is useful for inducing subjects to express expression from the nucleic acid of the invention. GAA polypeptides may be advantageous in terms of immune tolerance.

Other tissue-specific or non-tissue-specific promoters may also be useful in the practice of the invention. For example, the expression cassette may comprise a tissue-specific promoter that is different from a liver-specific promoter. For example, the promoter may be muscle specific, such as the desmin promoter (and desmin promoter variants such as the desmin promoter containing a natural or artificial enhancer), SPc5-12 or MCK promoter. In another embodiment, the promoter is a promoter specific for other cell lineages, such as an erythropoietin promoter, for expression of the GAA polypeptide from cells of the erythroid lineage.

In another embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include cytomegalovirus enhancer/chicken β-actin (CAG) promoter, cytomegalovirus enhancer/promoter (CMV), PGK promoter, SV40 early promoter, etc.

Furthermore, the promoter may also be an endogenous promoter, such as an albumin promoter or a GAA promoter.

In certain embodiments, the promoter is bound to an enhancer sequence such as a cis-acting regulatory module (CRM) or an artificial enhancer sequence. For example, the promoter can be bound to an enhancer sequence such as the human ApoE control region (or human apolipoprotein E/C-I locus, liver control region HCR-1 - Genbank accession number U32510, shown in SEQ ID NO: 15). In particular embodiments, enhancer sequences, such as ApoE sequences, are bound to liver-specific promoters, such as those listed above, in particular, for example, the hAAT promoter. Other CRMs useful in the practice of the present invention include those in Rincon et al. MolTher. 2015 Jan; 23(1):43-52; Chuah et al. MolTher. 2014 Sep; 22(9):1605-13 or Nair et al. Blood. 2014 May CRM as described in 15;123(20):3195-9.

In another specific embodiment, the nucleic acid construct comprises an intron, in particular an intron placed between the promoter and the GAA coding sequence. Introns can be introduced to improve mRNA stability and protein production. In other embodiments, the nucleic acid construct comprises a human beta-globin b2 (or HBB2) intron, a factor IX (FIX) intron, an SV40 intron, or a chicken beta-globin intron. In yet other embodiments, the nucleic acid constructs of the invention contain modified introns (especially modified HBB2 or FIX introns) designed to reduce the optional number of open reading frames (ARFs) or even complete removal of ARFs. Preferably, ARFs that span more than 50 bp in length and have a stop codon in-frame with the start codon are removed. ARF can be removed by modifying the sequence of the intron. For example, nucleotide substitutions, insertions or deletions may be utilized, preferably modification by nucleotide substitutions. As an example, one or more nucleotides, in particular one nucleotide, in an ATG or GTG start codon present in the intron sequence of interest can be replaced, creating a non-start codon. For example, the ATG or GTG within the target intron sequence can be replaced with a CTG that is not the start codon.

The classic HBB2 intron used in nucleic acid constructs is shown in SEQ ID NO:16. For example, the HBB2 intron can be modified by eliminating the initiation codon (ATG and GTG codons) in the intron. In a specific embodiment, the modified HBB2 intron comprised in the construct has the sequence set forth in SEQ ID NO: 17. The classic FIX intron used in nucleic acid constructs is derived from the first intron of human FIX and is shown in SEQ ID NO:18. The FIX intron can be modified by eliminating the start codon (ATG and GTG codons) in the intron. In a specific embodiment, the modified FIX intron comprised in the constructs of the invention has the sequence set forth in SEQ ID NO: 19. The classic chicken beta-globin intron used in nucleic acid constructs is shown in SEQ ID NO:20. The chicken beta-globin intron can be modified by eliminating the initiation codon (ATG and GTG codons) in the intron. In a specific embodiment, the modified chicken beta-globin intron comprised in the constructs of the invention has the sequence set forth in SEQ ID NO:21.

The inventors have previously shown in WO2015/162302 that this modified intron, especially the modified HBB2 or FIX intron, has advantageous properties and can significantly improve the expression of the transferred gene.

In a specific embodiment, the nucleic acid construct of the invention is an expression cassette comprising in a 5' to 3' direction a promoter optionally preceded by an enhancer, the coding sequence of the invention (i.e. the coding sequence of the invention An optimized truncated GAA coding sequence, a chimeric GAA coding sequence of the invention or a chimeric and sequence-optimized GAA coding sequence of the invention) and a polyadenylation signal (e.g., a bovine growth hormone polyadenylation signal, SV40 polyadenylation signal or another naturally occurring or artificial polyadenylation signal). In a specific embodiment, the nucleic acid construct of the invention is an expression cassette comprising a promoter, an intron, in a 5' to 3' orientation, optionally preceded by an enhancer (e.g., an ApoE control region) (especially an intron as defined above), the coding sequence of the invention and the polyadenylation signal. In another specific embodiment, the nucleic acid construct of the invention is an expression cassette comprising in the 5' to 3' direction an enhancer such as an ApoE control region, a promoter, an intron (in particular as defined above intron), the coding sequence of the invention and the polyadenylation signal. In other specific embodiments of the invention, the expression cassette comprises in the 5' to 3' direction an ApoE control region, a hAAT-liver-specific promoter, an HBB2 intron (especially a modified HBB2 intron as defined above). intron), the coding sequence of the invention and the bovine growth hormone polyadenylation signal, for example the construct shown in the following sequence:

- SEQ ID NO: 22, comprising an unoptimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 5;

- SEQ ID NO: 23, which includes a Δ8 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 1 Optimized sequence of signal peptide of NO:5;

- SEQ ID NO: 24, which includes a Δ8 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 1 Another optimized sequence of the signal peptide of NO:5;

- SEQ ID NO: 25, which includes a Δ8 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and SEQ ID NO : Optimized sequence of signal peptide 3;

- SEQ ID NO: 26, which includes the Δ42 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and SEQ ID NO : Optimized sequence of signal peptide 3;

- SEQ ID NO: 37, which includes a Δ29 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the unoptimized sequence of SEQ ID NO: 9) and SEQ ID NO: 1 The unoptimized sequence of the signal peptide of NO:3;

- SEQ ID NO: 38, which includes a Δ29 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and SEQ ID NO : Optimized sequence of signal peptide 3;

- SEQ ID NO: 39, which includes the Δ29 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and SEQ ID NO : Another optimized sequence of the signal peptide of 3;

- SEQ ID NO: 40, which includes the Δ42 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (the nucleotide sequence derived from the unoptimized sequence of SEQ ID NO: 9) and SEQ ID NO: 1 The unoptimized sequence of the signal peptide of NO:3;

- SEQ ID NO: 41, which includes the Δ42 truncated form of GAA encoding the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and SEQ ID NO : Another optimized sequence of the signal peptide of 3;

- SEQ ID NO: 42, which includes a Δ43 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the unoptimized sequence of SEQ ID NO: 9) and SEQ ID NO: 1 The unoptimized sequence of the signal peptide of NO:3;

- SEQ ID NO: 43, which includes a Δ43 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and SEQ ID NO : The optimized sequence of the signal peptide of 3; and

- SEQ ID NO: 44, which includes a Δ43 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and SEQ ID NO : Another optimized sequence of the signal peptide of 3;

- SEQ ID NO: 45, which includes a Δ47 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the unoptimized sequence of SEQ ID NO: 9) and SEQ ID NO: 1 The unoptimized sequence of the signal peptide of NO:3;

- SEQ ID NO: 46, which includes a Δ47 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and SEQ ID NO : The optimized sequence of the signal peptide of 3; and

- SEQ ID NO: 47, which includes a Δ47 truncated form encoding GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and SEQ ID NO :Another optimized sequence of the signal peptide of 3.

Other expression cassettes of the invention may include the following nucleic acid sequences:

- an unoptimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide.

In alternative embodiments of these particular constructs, the sequence encoding SEQ ID NO:1 is replaced by the sequence encoding SEQ ID NO:33.

In specific embodiments, the expression cassette contains an ApoE control region, a hAAT-liver-specific promoter, a codon-optimized HBB2 intron, the coding sequence of the invention, and a bovine growth hormone polyadenylation signal.

When designing nucleic acid constructs of the invention, those skilled in the art should be aware of the size limitations of vectors used to deliver the constructs to cells and organs. Specifically, those skilled in the art know that a major limitation of an AAV vector is its carrying capacity, which may vary with AAV serotype but is thought to be limited to around the size of the parent viral genome. For example, 5 kb is generally considered to be the maximum size packaged in AAV8 capsids (Wu Z. et al., Mol Ther., 2010, 18(1):80-86; Lai Y. et al., Mol Ther., 2010, 18(1) ):75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4):225-33). Therefore, those skilled in the art should take care in the practice of the present invention to select the components of the nucleic acid construct of the present invention so that the resulting nucleic acid sequence, including the sequence encoding the AAV 5'- to 3'-ITR, preferably does not exceed 110% of the carrying capacity of the AAV vector used, specifically preferably no more than 5.5 kb.

The invention also relates to vectors comprising the nucleic acid molecules or constructs disclosed herein. In particular, the vector of the present invention is a vector suitable for protein expression, preferably for gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing a nucleic acid molecule of the invention, in particular messenger RNA encoding a GAA polypeptide of the invention. In another embodiment, the vector is a transposon-based system that allows integration of the nucleic acid molecules or constructs of the invention into the genome of the target cell, such as the hyperactive Sleeping Beauty (SB100X) transposon seat system (Mates et al., 2009). In another embodiment, the vector is a viral vector suitable for gene therapy targeting any target cell such as liver tissue or cells, myocytes, CNS cells (e.g. brain cells) or hematopoietic stem cells such as cells of the erythroid lineage (e.g. red blood cells). In this case, the nucleic acid constructs of the invention also contain sequences known in the art that are suitable for the production of efficient viral vectors. In specific embodiments, the viral vector is derived from an integrating virus. In particular, the viral vectors may be derived from retroviruses or lentiviruses. In another specific embodiment, the viral vector is an AAV vector, such as an AAV vector suitable for transduction of liver tissue or cells, more particularly AAV-1, AAV-2 and AAV-2 variants (e.g. containing Y44 +500+730F+T491V modified quadruple mutated capsid-optimized AAV-2 engineered capsid, which was disclosed in Ling et al., 2016Jul 18, Hum Gene TherMethods. [Epub ahead of print]), AAV-3 and AAV-3 variants (such as the AAV3-ST variant comprising an engineered AAV3 capsid with two amino acid changes S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p .1042), AAV-3B and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-6 variants (e.g. AAV6 variants containing triple mutations in the Y731F/Y705F/T492V form of the AAV6 capsid entities, which are disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026), AAV-7, AAV-8, AAV-9, AAV-10 such as AAV-cy10 and AAV-rh10, AAV- Vectors such as rh74, AAV-dj, Anc80, LK03, AAV2i8, porcine AAV serotypes such as AAVpo4 and AAVpo6, or retroviral vectors such as lentiviral vectors and alpha-retroviruses. As is known in the art, depending on the particular viral vector contemplated for use, other suitable sequences can be introduced into the nucleic acid constructs of the invention for obtaining functional viral vectors. Suitable sequences include AAV ITR for AAV vectors or LTR for lentiviral vectors. Therefore, the invention also relates to an expression cassette as described above, carrying an ITR or LTR on each side.

Advantages of viral vectors are discussed in the remainder of this disclosure. Viral vectors are preferred for delivery of the nucleic acid molecules or constructs of the invention, such as retroviral vectors such as lentiviral vectors, or non-pathogenic parvoviruses, more preferably AAV vectors. The human parvovirus adeno-associated virus (AAV) is a naturally replication-deficient, dependent virus capable of integrating into the genome of infected cells to establish latent infection. This last property appears to be unique among mammalian viruses because the integration occurs at a specific site in the human genome called AAVS1, located on chromosome 19 (19q13.3-qter).

Therefore, AAV vectors have attracted considerable interest as potential vectors for human gene therapy. Advantageous properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines originating from different tissues that can be infected.

Among the serotypes of AAV isolated and well characterized from humans or non-human primates (NHP), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other currently used AAV serotypes include AAV -1. AAV-2 variants (e.g., capsid-optimized AAV-2 containing quadruple mutations of engineered capsids with Y44+500+730F+T491V changes, which was disclosed in Ling et al., 2016Jul 18, Hum Gene Ther Methods. [Epub ahead of print]), AAV-3, and AAV-3 variants (e.g., AAV3-ST variants containing an engineered AAV3 capsid with two amino acid changes S663V+T492V, disclosed in Vercauteren et al., 2016, Mol.Ther.Vol.24(6), p.1042), AAV-3B and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-6 variants (e.g. containing triple AAV6 variants in the mutated AAV6 capsid Y731F/Y705F/T492V form as disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026), AAV-7, AAV-8, AAV-9, AAV-10 such as AAV-cy10 and AAV-rh10, AAV-rh74, AAV-dj, Anc80, LK03, AAV2i8, porcine AAV serotypes such as AAVpo4 and AAVpo6, and tyrosine, lysine and serine coats of AAV serotypes Shell mutants, etc. Additionally, other non-natural engineered variants and chimeric AAVs may also be useful.

AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for modulating stability and particle lifetime, for high efficiency Degradation for precise delivery to the nucleus.

Ideal AAV fragments for assembly into vectors include the cap protein, including vp1, vp2, vp3, and the hypervariable region, the rep protein, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments can be readily used in a variety of different vector systems and host cells.

AAV-based recombinant vectors lacking the Rep protein integrate into the host genome with low efficiency and exist primarily as stable circular episomes that can persist in target cells for years.

In addition to using native serotypes of AAV, artificial AAV serotypes may also be used in the context of the present invention, including but not limited to AAV with non-naturally occurring capsid proteins. Such artificial capsids can be produced by any suitable technique using selected AAV sequences (e.g. fragments of the vp1 capsid protein) and can be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, non-AAV viruses Produced by combining heterologous sequences obtained from viral or non-viral sources. Artificial AAV serotypes may be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

Accordingly, the present invention relates to AAV vectors comprising the nucleic acid molecules or constructs of the invention. In the context of the present invention, the AAV vector contains an AAV capsid capable of transducing target cells of interest, in particular hepatocytes. According to a specific embodiment, the AAV vector has AAV-1, AAV-2, AAV-2 variants (e.g., a capsid-optimized AAV comprising a quadruple mutation of an engineered capsid with Y44+500+730F+T491V changes -2, which is disclosed in Ling et al., 2016Jul 18, Hum Gene TherMethods. [Epub ahead of print]), AAV-3 and AAV-3 variants (e.g., an engineered AAV3 coat containing two amino acid changes S663V+T492V AAV3-ST variant of the shell, which is disclosed in Vercauteren et al., 2016, Mol.Ther.Vol.24(6), p.1042), AAV-3B and AAV-3B variants, AAV-4, AAV-5 , AAV-6, and AAV-6 variants (e.g., AAV6 variants containing triple mutated AAV6 capsid Y731F/Y705F/T492V forms, disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026 ), AAV-7, AAV-8, AAV-9, AAV-10 such as AAV-cy10 and AAV-rh10, AAV-rh74, AAV-dj, Anc80, LK03, AAV2i8, porcine AAV such as AAVpo4 and AAVpo6, and AAV sera Serotypes of tyrosine, lysine, and serine capsid mutants. In specific embodiments, the AAV vector is of the AAV8, AAV9, AAVrh74, or AAV2i8 serotype (i.e., the AAV vector has a capsid of the AAV8, AAV9, AAVrh74, or AAV2i8 serotype). In another specific embodiment, the AAV vector is a pseudotyped vector, ie, its genome and capsid are derived from a different serotype of AAV. For example, the pseudotyped AAV vector may be one in which the genome is derived from one of the serotypes mentioned above and the capsid is derived from another serotype. For example, the genome of the pseudotyped vector can have a capsid derived from the AAV8, AAV9, AAVrh74 or AAV2i8 serotypes, and its genome can be derived from a different serotype. In a specific embodiment, the AAV vector has a capsid of the AAV8, AAV9 or AAVrh74 serotype, particularly the AAV8 or AAV9 serotype, more particularly the AAV8 serotype.

In specific embodiments, where the vector is used to deliver transgenes to muscle cells, the AAV vector may be selected from the group consisting of AAV8, AAV9, AAVrh74, and the like.

In another specific embodiment, where the vector is used to deliver transgenes to hepatocytes, the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80, AAV3B, etc. .

In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins. shell.

In a specific embodiment, the AAV vector is a chimeric vector, i.e., its capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains at least one VP capsid protein that would be derived from at least two AAV serotypes. Chimeric VP proteins based on VP protein regions or domain combinations of serotypes. Examples of such chimeric AAV vectors that can be used to transduce hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector may be derived from a combination of AAV8 capsid sequences and sequences from an AAV serotype that is different from the AAV8 serotype, such as any of the AAV serotypes specifically mentioned above. In another embodiment, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those disclosed in WO2015013313, in particular RHM4-1, RHM15 which exhibit high liver tropism -1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6 capsid variants.

In another embodiment, the modified capsids may also be derived from capsid modifications inserted by error-prone PCR and/or peptide insertion (eg as described in Bartel et al., 2011). Additionally, capsid variants may include single amino acid changes such as tyrosine mutants (eg as described in Zhong et al., 2008).

Furthermore, the genome of the AAV vector may be single-stranded or self-complementary, double-stranded (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal melting site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicated genomes are half the length of wild-type AAV genomes, have a tendency to package DNA dimers. In preferred embodiments, the AAV vectors used in the practice of the invention have a single-stranded genome, and more preferably comprise an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, particularly an AAV8, AAV9 or AAVrh74 capsid such as an AAV8 or AAV9 capsid. capsids, more specifically AAV8 capsids.

In a particularly preferred embodiment, the invention relates to an AAV vector comprising a nucleic acid construct of the invention in a single- or double-stranded self-complementary genome (eg a single-stranded genome). In one embodiment, the AAV vector comprises an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, particularly an AAV8, AAV9 or AAVrh74 capsid such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. In another specific embodiment, the nucleic acid is operably linked to a promoter, in particular a ubiquitous or liver-specific promoter. According to specific variant embodiments, the promoter is a ubiquitous promoter, such as cytomegalovirus enhancer/chicken beta-actin (CAG) promoter, cytomegalovirus enhancer/promoter (CMV), PGK promoter and SV40 early promoter. In certain variants, the ubiquitous promoter is the CAG promoter. According to another variant, said promoter is a liver-specific promoter, such as alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter and thyroxine-binding globulin ( TBG) promoter. In certain variants, the liver-specific promoter is the hAAT liver-specific promoter of SEQ ID NO:14. In another specific embodiment, the nucleic acid construct contained in the genome of the AAV vector of the present invention also contains an intron as described above, for example, placed between the promoter and the encoding GAA coding sequence (i.e., the optimization of the present invention The intron between the nucleic acid sequence of the GAA coding sequence, the chimeric GAA coding sequence of the invention or the chimeric and optimized GAA coding sequence of the invention). Representative introns that may be included within nucleic acid constructs introduced into the AAV vector include, but are not limited to, human beta-globin b2 (or HBB2) intron, FIX intron, and chicken beta-globin intron. son. The introns in the genome of the AAV vector may be classical (or unmodified) introns or designed to reduce the number of alternative open reading frames (ARFs) in the introns or even completely Removing the modified intron of ARF. Modified and unmodified introns that may be used in the practice of this embodiment of introducing the nucleic acid of the invention into an AAV vector have been fully described above. In particular embodiments, the AAV vectors of the invention, in particular AAV vectors comprising AAV8, AAV9, AAVrh74 or AAV2i8 capsids, in particular AAV8, AAV9 or AAVrh74 capsids such as AAV8 or AAV9 capsids, more particularly AAV8 capsids , including modified (or optimized) introns in its genome, such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19 and the modification of SEQ ID NO: 21 The chicken β-globin intron. In another specific embodiment, the vector of the invention is an AAV vector comprising an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid, It contains a genome containing in 5' to 3' direction: AAV 5'-ITR (e.g. AAV2 5'-ITR), ApoE control region, hAAT-liver-specific promoter, HBB2 intron (in particular as defined above Modified HBB2 intron), the GAA coding sequence of the invention, the bovine growth hormone polyadenylation signal and the AAV 3'-ITR (e.g., AAV2 3'-ITR), for example, the genome is contained in SEQ ID NO: 22 to 26 and the nucleic acid constructs shown in SEQ ID NOs: 37 to 47, and flanked by an AAV 5'-ITR (eg, AAV2 5'-ITR) and an AAV 3'-ITR (eg, AAV2 3'-ITR). Other nucleic acid constructs useful in the practice of the invention include those described above, including:

- an unoptimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an unoptimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding the signal peptide of SEQ ID NO: 4 or 6 The optimized nucleotide sequence;

- encoding a Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide;

- encoding a Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding SEQ ID NO: 4, 6 or 7 Optimized nucleotide sequence of signal peptide.

In alternative embodiments of these particular constructs, the sequence encoding SEQ ID NO:1 is replaced by the sequence encoding SEQ ID NO:33.

In a specific embodiment of the invention, the nucleic acid construct of the invention comprises a liver-specific promoter as defined above, and the vector is a viral vector capable of transducing liver tissue or cells as described above. The inventors present data below showing that thanks to this embodiment a highly efficient and optimized vector was developed to express a highly secretable form of GAA in hepatocytes and induce immune tolerance to the protein, pro-tolerance of the liver Protochemical and metabolic properties are advantageously achieved.

Furthermore, in another specific embodiment, the present invention provides a combination of two vectors, such as two viral vectors, in particular two AAV vectors, for improved gene delivery and therapeutic efficacy in target cells. For example, the two vectors may carry a nucleic acid molecule of the invention encoding a GAA protein of the invention and under the control of a different promoter in each of the two vectors. In a specific embodiment, one vector contains a promoter that is a liver-specific promoter (such as one described above) and the other vector contains a promoter specific for another target tissue for the treatment of glycogen storage disease. Specific promoters include muscle-specific promoters, such as the desmin promoter. In a specific variation of this embodiment, this combination of vectors corresponds to multiple co-packaged AAV vectors generated as described in WO2015196179.

The invention also relates to cells, such as hepatocytes, transformed with the nucleic acid molecules or constructs of the invention, as is the case for ex vivo gene therapy. The cells of the invention may be delivered to a subject in need thereof, such as a GAA-deficient patient, by any suitable route of administration, for example by injection into the subject's liver or bloodstream. In a specific embodiment, the present invention includes introducing the nucleic acid molecule, nucleic acid construct or vector of the invention, especially the lentiviral vector, into hepatocytes, especially the hepatocytes of the subject to be treated, and introducing therein said The transformed hepatocytes of the nucleic acid are administered to the subject. Advantageously, this embodiment is useful for secreting GAA from said cells. In specific embodiments, the hepatocytes are hepatocytes from the patient to be treated, or are liver stem cells that are further transformed and differentiated into hepatocytes in vitro for subsequent administration to the patient.

The invention also relates to a transgenic non-human animal comprising in its genome a nucleic acid molecule or construct encoding a GAA polypeptide of the invention. In specific embodiments, the animal is a mouse.

In addition to the specific delivery systems presented in the examples below, a variety of different delivery systems are known and can be used to administer the nucleic acid molecules or constructs of the invention, e.g. encapsulated in liposomes, microparticles, microparticles Capsules, recombinant cells capable of expressing the coding sequences of the invention, receptor-mediated endocytosis, constructing therapeutic nucleic acids as part of a retrovirus or other vector, etc.

According to one embodiment, it may be desired to introduce a GAA polypeptide, nucleic acid molecule, nucleic acid construct or cell of the invention into the liver of the subject via any suitable route. In addition to naked DNA, minicircles and transposons, for example, can be used for lentiviral vector delivery. In addition, gene editing technologies such as zinc finger nucleases, meganucleases, TALENs and CRISPR can also be used to deliver the coding sequences of the invention.

The invention also provides pharmaceutical compositions comprising the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention. These compositions comprise a therapeutically effective amount of the therapeutic agent (nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention) and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States or European Pharmacopoeia or other recognized pharmacopeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or medium with which the therapeutic agent is administered. These pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc.

If desired, the compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin. These compositions should contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, and a suitable amount of carrier so as to provide a form suitable for administration to the subject. In specific embodiments, the nucleic acids, vectors or cells of the invention are formulated in a composition comprising phosphate buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acids, vectors or cells of the invention are formulated in a solution containing Ringer's lactate solution and a final concentration of non-ionic acid at a final concentration of 0.01-0.0001%, e.g. a concentration of 0.001% by weight of the total composition. surfactant such as pluronic F68. The composition may also comprise serum albumin, in particular human serum albumin, for example 0.25% human serum albumin. Other formulations suitable for storage or administration are known in the art, inter alia from WO 2005/118792 or Allay et al., 2011.

In preferred embodiments, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also contain solubilizers and local anesthetics such as lignocaine to relieve pain at the injection site.

In one embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention can be delivered in vesicles, especially liposomes. In another embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention can be delivered in a controlled release system.

Methods of administration of the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. In specific embodiments, the administration is by intravenous or intramuscular route. The nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention, whether vectored or not, may be administered by any convenient route, such as by infusion or bolus injection, by transepithelial or mucosal linings (e.g. oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other bioactive agents. Administration may be systemic or local.

In certain embodiments, it may be desirable to administer pharmaceutical compositions of the present invention topically to the area in need of treatment, such as the liver. This can be achieved, for example, using implants that are porous, non-porous or gel-like materials, including membranes such as silicone rubber membranes or fibers.

The amount of a therapeutic agent of the invention (ie, a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention) that is effective in the treatment of glycogen storage disease can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be used to aid in predicting optimal dosage ranges. The precise dosage used in the formulations will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The dosage of the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention administered to a subject in need thereof will vary depending on several factors, including, but not limited to, the route of administration, the specific disease being treated, the condition of the subject age or the level of expression necessary to achieve a therapeutic effect. Those skilled in the art can readily determine the desired dosage range based on these and other factors based on knowledge in the art. Where the treatment involves administering to the subject a viral vector, such as an AAV vector, a typical dosage of the vector is at least ^1x10 vector genomes per kilogram of body weight (vg/kg), for example at least ^1x10 vg/kg, at least ^1x10 vg /kg, at least 1x10 ¹¹ vg/kg, at least 1x10 ¹² vg/kg, at least 1x10 ¹³ vg/kg or at least 1x10 ¹⁴ vg/kg.

The invention also relates to a method for treating glycogen storage disease, said method comprising the step of delivering a therapeutically effective amount of a nucleic acid, vector, GAA polypeptide, pharmaceutical composition or cell of the invention to a subject in need thereof.

The present invention also relates to a method for treating glycogen storage disease, which method does not induce an immune response against the transferred gene (ie, against the GAA polypeptide of the invention), or induces an immune response against the transferred gene. Reduced immune response, the method comprising the step of delivering a therapeutically effective amount of a nucleic acid molecule, nucleic acid construct, vector, pharmaceutical composition or cell of the invention to a subject in need thereof. The present invention also relates to a method for treating glycogen storage disease, said method comprising repeatedly administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule, nucleic acid construct, vector, pharmaceutical composition or cell of the invention. In this case, the nucleic acid molecule or nucleic acid construct of the invention contains a promoter that is functional in hepatocytes, thereby allowing immune tolerance to the expressed GAA polypeptide to be generated therefrom. Likewise, in this case, the pharmaceutical composition used in this case contains a nucleic acid molecule or a nucleic acid construct that contains a promoter that is functional in hepatocytes. In the case of delivering hepatocytes, the cells may have been previously collected from the subject in need of treatment and engineered by introducing therein a nucleic acid molecule or nucleic acid construct of the invention such that they are capable of producing a GAA polypeptide of the invention. Cell. According to one embodiment, where repeated administration is involved, said administration may be repeated at least once or more, and may even be considered to occur on a regular schedule, such as weekly, monthly or yearly. The regular schedule may also include dosing every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or more. In another specific embodiment, each administration of a viral vector of the invention is performed using a different virus for each successive administration, thereby avoiding possible immune responses to previously administered viral vectors. resulting in reduced performance. For example, a viral vector containing AAV8 capsids may be administered first, followed by a vector containing AAV9 capsids or even a virus unrelated to AAV such as a retroviral or lentiviral vector.

According to the present invention, treatment may include curative, palliative or preventive effects. Accordingly, therapeutic and preventive treatments include amelioration of symptoms of a particular glycogen storage disease or prevention of or otherwise reducing the risk of developing a particular glycogen storage disease. The term "preventative" may be thought of as reducing the severity or incidence of a particular condition. "Prophylactic" also includes preventing the recurrence of a particular condition in a patient previously diagnosed with that condition. "Therapeutic" can also reduce the severity of existing conditions. The term "treatment" is used herein to refer to any regimen that may benefit an animal, particularly a mammal, and more particularly a human subject.

The present invention also relates to an ex vivo gene therapy method for treating glycogen storage diseases, said method comprising introducing the nucleic acid molecule or nucleic acid construct of the invention into isolated cells, such as isolated hematopoietic stem cells, of a patient in need of treatment. and introducing the cells into the patient in need of treatment. In a specific embodiment of this case, said nucleic acid molecule or construct is introduced into said cell using a vector as defined above. In specific embodiments, the vector is an integrating viral vector. In another specific embodiment, the viral vector is a retroviral vector such as a lentiviral vector. For example, the lentiviral vectors disclosed in van Til et al., 2010, Blood, 115(26), p. 5329 can be used in the practice of the methods of the invention.

The invention also relates to a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use as a medicament.

The invention also relates to the nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use in a method for treating diseases caused by mutations in the GAA gene, in particular for use in methods for treating Pompe disease. . The invention also relates to the nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use in a method of treating a glycogen storage disease, such as GSDI (von Gierke's disease), GSDII (Pompe disease), GSDIII (Corey's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage diseases of the heart, more particularly GSDI, GSDII or GSDIII, Even more specifically GSDII and GSDIII, most specifically GSDII. Truncated GAA polypeptides of the invention may be administered to a patient in need thereof for use in enzyme replacement therapy (ERT), for example for glycogen storage diseases such as GSDI (Von Gierke's disease), GSDII (Pompe's disease), GSDIII (Corrie's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage diseases of the heart, more particularly GSDI, GSDII or GSDIII, even more particularly GSDII and GSDIII, most particularly GSDII.

The present invention also relates to the use of the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the present invention in the manufacture of medicaments, which can be used to treat glycogen storage diseases such as GSDI (von Gierke's disease), GSDII (Pompe's disease), GSDIII (Corrie's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage diseases of the heart, more particularly GSDI, GSDII or GSDIII, even more particularly GSDII and GSDIII, most especially GSDII.

Example

The invention is described in more detail with reference to the following experimental examples and drawings. These examples are provided for illustrative purposes only and are not intended to be limiting.

Materials and methods

GAA activity

GAA activity was measured after homogenizing frozen tissue samples in distilled water. Weigh out 50-100mg of tissue and homogenize, then centrifuge at 10,000xg for 20 minutes. In a 96-well plate, set up the reaction using 10 μl of supernatant and 20 μl of substrate 4MU α-D-glucoside. The reaction mixture was incubated at 37°C for 1 hour and then stopped by adding 150 μl of sodium carbonate buffer pH 10.5. Fluorescent 4MU released from each reaction mixture was measured using an EnSpire alpha plate reader (Perkin-Elmer) at 449 nm (emission) and 360 nm (excitation) using a standard curve (0-2500 pmol/μl of 4MU). The protein concentration of the clarified supernatant was quantified by BCA (Thermo Fisher Scientific). To calculate GAA activity, divide the released 4MU concentration by the protein concentration of the sample and report the activity in nmol/hour/mg protein.

mouse research

Gaa-/- mice were generated by targeted disruption of exon 6 and maintained on a C57BL/6J/129X1/SvJ background (Raben N. et al., 1998). The vector was delivered via the tail vein in a volume of 0.2 ml. Serum samples were collected monthly to monitor levels of secreted hGAA. PBS-injected diseased animals and wild-type littermates were used as controls.

NHP research

Male cynomolgus monkeys were housed in stainless steel cages and maintained under a 12-h light/dark cycle. All cynomolgus monkeys had neutralizing antibody titers <1:5 before starting the study. A dose of 2E12 vg/kg of AAV8-hAAT-sp7-Δ8-hGAAco1 was infused via the saphenous vein. Blood samples were obtained via the femoral vein 12 days before and 30 days after the injection. Whole blood was collected in EDTA-containing tubes and centrifuged to separate serum. Three months after vehicle administration, all cynomolgus monkeys were euthanized. The animals were first anesthetized with a ketamine/dexmedetomidine mixture and then euthanized using an IV injection of sodium pentobarbital. Tissues were collected immediately and frozen in liquid nitrogen.

Western blot analysis

Total homogenates were obtained from frozen muscles. Protein concentration in the extracts was determined by Pierce BCA protein assay (Thermo Fisher Scientific) following the manufacturer's instructions. Western blotting was performed using anti-hGAA antibody (Abcam). Anti-tubulin antibody (Sigma Aldrich) was used as a loading control.

result

With the aim of designing new forms of GAA with improved secretion and reduced immunogenicity, we decided to generate truncated forms of GAA and optionally combine them with alternative signal peptides.

The human GAA shown in SEQ ID NO:2 served as the basis for designing these new forms. SEQ ID NO: 1 corresponds to the sequence of SEQ ID NO: 2, but does not contain the corresponding native signal peptide of GAA (amino acids 1-27 of SEQ ID NO: 2). A nucleic acid construct was designed to encode a GAA polypeptide derived from SEQ ID NO: 1 truncated at the N-terminal end. We designed deletions on the basis of the wild-type hGAA coding sequence (SEQ ID NO:9, which corresponds to nucleotides 82-2859 of SEQ ID NO:8 as the wild-type hGAA coding sequence including the signal peptide coding sequence) Beginning with the nucleic acid sequence (Δ8) corresponding to the codons for the first 8 amino acids of SEQ ID NO:1. In addition to the wild-type hGAA coding sequence, we also designed optimized nucleic acid sequences encoding the Δ8 truncated hGAA polypeptide (SEQ ID NO: 10 and SEQ ID NO: 11 correspond to the optimized coding sequences of hGAAco1 and hGAAco2, respectively) , to rule out possible sequence-specific effects.

Table 1. Description of optimized sequences. This table illustrates the comparison of the characteristics of the two hGAA optimized sequences with those of the wild type. a) Codon adaptation index and b) GC content were calculated using the rare codon analysis tool (http://www.genscript.com). c) and d) are the alternative open reading frames calculated on the 5’ to 3’ (aORF 5’→3’) and 3’ to 5’ (aORF 3’→5’) strands, respectively. e) and f) are the acceptor (SA) and donor (SD) splice sites calculated using the splice site online prediction tool (http://www.fruitfly.org/seq_tools/splice.html), respectively. g) and h) are percent identities calculated for the wild-type (wt) and optimized co1 sequences, respectively. i) CpG islands were calculated using the MethDB online tool (http://www.methdb.de/links.html). CpG islands are sequences longer than 100 bp, with a GC content >60% and an observed/expected ratio >0.6.

Amino acids 1-27 of the hGAA sequence (corresponding to the native signal peptide of hGAA, defined here as sp1; its sequence is shown in SEQ ID NO: 4) were used with human α-1-, defined here as sp2. Amino acids 1-24 of the sequence of antitrypsin (NP_000286.3) (sequence shown in SEQ ID NO: 5) were substituted. We transfected constructs encoding truncated hGAA into human hepatoma cells (Huh-7) in parallel with their full-size versions and measured the amount of hGAA released in the culture medium after 48 h (Fig. 1A). The Δ8 deletion of the hGAA sequence resulted in a significant, 50% increase in secretion levels for both wild-type (hGAA) and codon-optimized (hGAAco2) sequences. The same truncation performed on a different codon-optimized sequence (hGAAco1) also enhanced hGAA secretion to the same extent.

To confirm that changes in the sequence after the signal peptide can enhance hGAA secretion, we further truncated the hGAA polypeptide. We eliminated the codons corresponding to the first 42 amino acids of hGAA (Δ42) from the hGAAco1 construct and replaced them with a signal peptide derived from chymotrypsinogen B1 (sp7; sequence shown in SEQ ID NO: 3 middle) instead. We then compared the efficacy of the secretion obtained using this new deleted construct with that using the Δ8 version fused to the sp7 signal peptide and the full-size hGAAco1 with sp1 or sp7. We transfected these constructs into Huh-7 cells and measured hGAA activity in the culture medium 48 hours after transfection. As expected, we could measure hGAA activity after transfection of full-size hGAAco1 (p=0.055 vs. GFP), and by replacing the wild-type signal peptide with sp7, its secretion was improved 2-fold (p=0.006 vs. hGAAco1 ). Surprisingly, both the Δ8 and Δ42 hGAA sequences fused to the sp7 signal peptide showed a 2-fold increase in secreted hGAA compared to the full-size sequence (p=0.0002 and 0.0003, respectively, relative to sp7-hGAAco1, Figure 1B).

Taken together, these data demonstrate that truncation of the hGAA sequence combined with a highly efficient signal peptide can enhance secretion of the protein in vitro. Furthermore, the truncation has an important advantage over mutations of the native sequence in that it does not generate large amounts of neoantigens, which is an advantage in the engineering of therapeutic products.

We then validated these findings in vivo, in a mouse model of Pompe disease. We injected GAA-/- mice (Raben et al., J. Bio. Chem. 1998) with an AAV8 vector derived from the apolipoprotein B enhancer and the human alpha-1-antitrypsin promoter. Full-size, Δ8 or Δ42 hGAAco1 fused to the sp7 signal peptide is expressed under the transcriptional control of the fusion's high-strength liver-specific promoter (hAAT). One month after injection of 2E12 vg/kg of vector as described above, mice were bled and hGAA activity in serum was measured. Mice treated with a vector expressing full-length hGAAco1 fused to sp7 showed increased hGAA levels in the bloodstream (p=0.115 vs. PBS). Surprisingly, both truncated hGAA Δ8 and Δ42 caused significant increases in hGAA levels in serum (p=0.014 and 0.013, respectively).

These data indicate that deletion of the front amino acid of hGAA causes a significant increase in the levels of secreted hGAA in the bloodstream.

Additionally, another signal peptide corresponding to amino acids 1-25 of iduronate-2-sulfatase (sp6; SEQ ID NO: 6) was fused to the Δ8 truncated form of hGAA. In parallel with plasmids expressing optimized hGAA (hGAAco1) fused to sp1, sp2, sp6, sp7 or sp8, we used plasmids expressing GFP or wild-type hGAA (hGAA; corresponding to amino acid residues 28-952 of SEQ ID NO: 30 Hepatoma cells (Huh-7) were transfected with a plasmid based on the parent polypeptide). At 48 hours after transfection, the growth medium was analyzed for the presence of hGAA. Notably, these constructs caused hGAA secretion levels that were significantly higher than those observed in the negative control represented by GFP-transfected cells (Fig. 3).

We then assessed glycogen content in the heart, diaphragm, and quadriceps muscles of GAA-/- mice treated with Δ8-hGAA as described above. Notably, we observed high levels of hGAA in tissues after treatment with the Δ8-hGAAco expression vector (data not shown), which correlated with a significant decrease in glycogen content in all tissues considered (Figure 4B- D). Specifically, in the heart (Fig. 4B), glycogen levels measured after treatment with vehicle bearing efficient signal peptides sp7 and 8 were indistinguishable from those observed in undiseased animals (relative to WT p=0.983 and 0.996 respectively). Importantly, the levels observed after treatment with both sp7 and sp8 vectors were significantly reduced compared to GAA-/- animals injected with PBS or treated with the hGAAco expression vector fused to the sp1 signal peptide.

We also tested whether hepatic transduction using our vector induces a humoral response to the introduced gene. Mice were injected intravenously with AAV8 vectors expressing hGAAco1(co) with the native sp1 signal peptide or Δ8-hGAAco1 fused to sp2, sp7, or sp8 under the transcriptional control of a liver-specific promoter. The results are presented in Figure 5. Gaa-/- injected intramuscularly with AAV expressing Δ8-hGAAco1 under the transcriptional control of a constitutive promoter showed very high levels of total IgG (∼150 μg/mL), whereas vectors expressing the same protein in the liver total showed lower humoral response levels. Interestingly, mice injected with a vector expressing sp1hGAAco1(co) showed detectable antibody levels at both doses, whereas mice injected with the engineered high-secretion vector had undetectable IgG levels. These data indicate that expression of the transgene in the liver is fundamental for the induction of peripheral tolerance and they also provide an indication that the high circulating hGAA levels achieved by fusion with a highly efficient signal peptide induces resistance to Reduction in humoral response to the protein itself.

The best-performing vector selected in the mouse study was injected into two non-human primates (NHP, cynomolgus monkeys) to verify the secretion potency and uptake into muscle of our vector. We injected two monkeys with 2E12vg/kg of AAV8-hAAT-sp7-Δ8-hGAAco1. One month after injection, we measured hGAA levels in the serum of both animals by Western blotting using specific anti-hGAA antibodies. We observed clear bands in two monkeys with a size consistent with hGAA. This band was absent in serum samples obtained 12 days before vector injection, confirming the specificity of our assay (Fig. 6A). We sacrificed the animals three months after injection and harvested tissues to confirm whether hGAA secreted from the liver into the bloodstream was efficiently taken up by the muscles. We performed Western blots of total lysates obtained from biceps and septum of two monkeys using antibodies specific for hGAA. Interestingly, we were able to observe clear bands in animal 2, which also showed the highest levels of hGAA in the bloodstream (Figure 6B). In animal 1, we could also observe weaker bands with molecular weights consistent with hGAA in both analyzed muscles. These data demonstrate that the AAV8-hAAT-sp7-Δ8-hGAAco1 vector efficiently transduces the liver in NHP. They also demonstrate that proteins secreted in the bloodstream are efficiently taken up into muscle, and that this uptake correlates with hGAA levels measured in the blood.

We also determined the effect of the vector selected for best performance in mouse studies (AAV8-hAAT-sp7-Δ8-hGAAco1) in the GSDIII mouse model. We developed a knockout mouse model of glycogen debranching enzyme (GDE). This model recapitulates the disease phenotype observed in humans with glycogen storage disease type III (GSDIII). Specifically, GDE-/- mice that completely lack GDE activity have impaired muscle strength and accumulate glycogen in different tissues. Interestingly, they also accumulate glycogen in the liver, which is also observed in humans. Here, we tested whether overexpression of sp7-Δ8-hGAA in the liver rescues the glycogen accumulation observed in GDE-/- mice. We injected GDE-/- mice with 1E11 or 1E12 vg/mouse of AAV8-hAAT-sp7-Δ8-hGAAco1. As controls, we injected wild-type (WT) and GDE-/- mice in parallel with PBS. Three months after vehicle administration, mice were sacrificed and glycogen levels in the liver were quantified. The results are reported in Figure 7. As already reported (Pagliarani et al. and our model), GDE-/- mice showed a significant increase in glycogen accumulation in the liver (p=1.3E-7), with 5-fold more when compared to wild-type animals. of glycogen. Surprisingly, treatment with 1E11 and 1E12 vg/mouse AAV8-hAAT-sp7-Δ8-hGAAco1 vector caused a statistically significant reduction in glycogen content (p=4.5E-5 and 1.4E-6, respectively ). Importantly, glycogen levels measured in the livers of mice injected with the AAV8-hAAT-sp7-Δ8-hGAAco1 vector were indistinguishable from those measured in wild-type animals, especially at the highest dose (for p=0.053 for the 1E11 dose cohort and 0.244 for the 1E12 dose cohort).

We performed analyzes of GAA activity in the culture medium and lysates of HuH7 cells transfected with different versions of GAA (all codon optimized): 1. native GAA(co) including the native sp1GAA signal peptide, 2. Engineered GAA with heterologous sp7 signal peptide (sp7-co), and 3. Engineered GAA containing heterologous sp7 signal peptide and then deleted different numbers of amino acids (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co and sp7-Δ62-co, in which the first 8, 29, 42, 47 and 62 N-terminal amino acids of SEQ ID NO: 1 are deleted respectively) . The analysis showed (Fig. 8) that in the culture medium of cells transfected with the Δ8, Δ29, Δ42 and Δ43 GAA versions compared to both engineered non-deleted GAA (sp7-co) and native GAA (co) GAA activity was significantly higher. In contrast, compared with other engineered GAA versions [deleted (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co) and non-deleted (sp7-co)], Significantly lower GAA activity was observed in the culture medium of cells transfected with the Δ47 and Δ62 GAA versions. Interestingly (Fig. 9) between productive deleted (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co) and non-deleted versions (sp7-co) cells There were no differences in intracellular GAA activity, indicating that they are both efficiently produced and processed within the cell. In contrast, intracellular GAA activity was very low for the sp7-Δ47-co and sp7-Δ62-co versions and was consistent with all other engineered versions [deleted (sp7-Δ8-co, sp7-Δ29-co, sp7 -Δ42-co, sp7-Δ43-co) and not deleted (sp7-co)] were significantly lower.

We also performed analyzes of GAA activity in the culture medium and lysates of HuH7 cells transfected with different versions of GAA (all codon optimized): 1. native GAA(co) including the native sp1GAA signal peptide, 2. Engineered GAA containing heterologous sp6 or sp8 signal peptide (sp6-co, sp8-co), and 3. Engineered GAA containing heterologous sp6 or sp8 signal peptide and then deleted 8 amino acids (sp6-Δ8- co, sp8-Δ8-co). The analysis showed (Figure 10) that, compared with i. their corresponding engineered non-deleted GAA versions (sp6-co or sp8-co) and ii. native GAA (co), the GAA activity was significantly higher in the cells' culture medium. Interestingly, there was no difference in intracellular GAA activity between all engineered versions of GAA (both deleted and non-deleted), indicating that they are all efficiently produced and processed within the cell (cell lysate plot). In contrast, intracellular GAA activity was significantly higher when native GAA(co) was used compared with the engineered version, indicating that native GAA was primarily retained within the cell.

sequence list

<110> Genezon Company, etc.

<120> Acid alpha-glucosidase variants and their uses

<130> B2299PC00

<160> 53

<170>PatentIn version 3.3

<210> 1

<211> 925

<212> PRT

<213> Artificial sequence

<220>

<223> hGAAwt w/o sp

<400> 1

Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser

1 5 10 15

Gly Ser Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln Gly

20 25 30

Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro

35 40 45

Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp

50 55 60

Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly

65 70 75 80

Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly

85 90 95

Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu

100 105 110

Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr

115 120 125

Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val

130 135 140

Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala

145 150 155 160

Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val His Ser Arg

165 170 175

Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly

180 185 190

Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr

195 200 205

Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser

210 215 220

Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu

225 230 235 240

Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu

245 250 255

Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu

260 265 270

Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser

275 280 285

Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg

290 295 300

Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro

305 310 315 320

Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met

325 330 335

Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser

340 345 350

Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His

355 360 365

Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg

370 375 380

Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met

385 390 395 400

Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp

405 410 415

Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp

420 425 430

Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro

435 440 445

Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr

450 455 460

Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His

465 470 475 480

Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser

485 490 495

Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu

500 505 510

Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala

515 520 525

Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu

530 535 540

His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu

545 550 555 560

Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe

565 570 575

Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser

580 585 590

Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn

595 600 605

Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly

610 615 620

Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe

625 630 635 640

Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu

645 650 655

Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu

660 665 670

Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln

675 680 685

Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe

690 695 700

Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly

705 710 715 720

Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val

725 730 735

Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro

740 745 750

Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu

755 760 765

Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu

770 775 780

Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln

785 790 795 800

Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu

805 810 815

Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp

820 825 830

Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln

835 840 845

Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg

850 855 860

Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu

865 870 875 880

Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val

885 890 895

Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val

900 905 910

Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

915 920 925

<210> 2

<211> 952

<212> PRT

<213> Homo sapiens

<400> 2

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 3

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> sp7

<400> 3

Met Ala Phe Leu Trp Leu Leu Ser Cys Trp Ala Leu Leu Gly Thr Thr

1 5 10 15

Phe Gly

<210> 4

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> sp1

<400> 4

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu

20 25

<210> 5

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> sp2

<400> 5

Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys

1 5 10 15

Cys Leu Val Pro Val Ser Leu Ala

20

<210> 6

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> sp6

<400> 6

Met Pro Pro Pro Arg Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val

1 5 10 15

Leu Ser Ser Val Cys Val Ala Leu Gly

20 25

<210> 7

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> sp8

<400> 7

Met Ala Ser Arg Leu Thr Leu Leu Thr Leu Leu Leu Leu Leu Leu Ala

1 5 10 15

Gly Asp Arg Ala Ser Ser

20

<210> 8

<211> 2859

<212> DNA

<213> Homo sapiens

<400> 8

atgggagtga ggcacccgcc ctgctcccac cggctcctgg ccgtctgcgc cctcgtgtcc 60

ttggcaaccg cagcgctcct ggggcacatc ctactccatg atttcctgct ggttccccga 120

gagctgagtg gctcctcccc agtcctggag gagactcacc cagctcacca gcagggagcc 180

agcagaccag ggccccggga tgcccaggca caccccgggc ggccgcgagc agtgcccaca 240

cagtgcgacg tcccccccaa cagccgcttc gattgcgccc ctgacaaggc catcacccag 300

gaacagtgcg aggcccgcgg ctgttgctac atccctgcaa agcaggggct gcagggagcc 360

cagatggggc agccctggtg cttcttccca cccagctacc ccagctacaa gctggagaac 420

ctgagctcct ctgaaatggg ctacacggcc accctgaccc gtaccacccc caccttcttc 480

cccaaggaca tcctgaccct gcggctggac gtgatgatgg agactgagaa ccgcctccac 540

ttcacgatca aagatccagc taacaggcgc tacgaggtgc ccttggagac cccgcatgtc 600

cacagccggg caccgtcccc actctacagc gtggagttct ccgaggagcc cttcggggtg 660

atcgtgcgcc ggcagctgga cggccgcgtg ctgctgaaca cgacggtggc gcccctgttc 720

tttgcggacc agttccttca gctgtccacc tcgctgccct cgcagtatat cacaggcctc 780

gccgagcacc tcagtcccct gatgctcagc accagctgga ccaggatcac cctgtggaac 840

cgggaccttg cgcccacgcc cggtgcgaac ctctacgggt ctcacccttt ctacctggcg 900

ctggaggacg gcgggtcggc acacggggtg ttcctgctaa acagcaatgc catggatgtg 960

gtcctgcagc cgagccctgc ccttagctgg aggtcgacag gtgggatcct ggatgtctac 1020

atcttcctgg gcccagcc caagagcgtg gtgcagcagt acctggacgt tgtgggatac 1080

ccgttcatgc cgccatactg gggcctgggc ttccacctgt gccgctgggg ctactcctcc 1140

accgctatca cccgccaggt ggtggagaac atgaccaggg cccacttccc cctggacgtc 1200

cagtggaacg acctggacta catggactcc cggagggact tcacgttcaa caaggatggc 1260

ttccgggact tcccggccat ggtgcaggag ctgcaccagg gcggccggcg ctacatgatg 1320

atcgtggatc ctgccatcag cagctcgggc cctgccggga gctacaggcc ctacgacgag 1380

ggtctgcgga ggggggtttt catcaccaac gagaccggcc agccgctgat tgggaaggta 1440

tggcccgggt ccactgcctt ccccgacttc accaacccca cagccctggc ctggtggggag 1500

gacatggtgg ctgagttcca tgaccaggtg cccttcgacg gcatgtggat tgacatgaac 1560

gagccttcca acttcatcag gggctctgag gacggctgcc ccaacaatga gctggagaac 1620

ccaccctacg tgcctggggt ggttgggggg accctccagg cggccaccat ctgtgcctcc 1680

agccaccagt ttctctccac acactacaac ctgcacaacc tctacggcct gaccgaagcc 1740

atcgcctccc acagggcgct ggtgaaggct cgggggacac gcccatttgt gatctcccgc 1800

tcgacctttg ctggccacgg ccgatacgcc ggccactgga cgggggacgt gtggagctcc 1860

tggggagcagc tcgcctcctc cgtgccagaa atcctgcagt ttaacctgct gggggtgcct 1920

ctggtcgggg ccgacgtctg cggcttcctg ggcaacacct cagaggagct gtgtgtgcgc 1980

tggacccagc tgggggcctt ctaccccttc atgcggaacc acaacagcct gctcagtctg 2040

ccccaggagc cgtacagctt cagcgagccg gcccagcagg ccatgaggaa ggccctcacc 2100

ctgcgctacg cactcctccc ccacctctac acactgttcc accaggccca cgtcgcgggg 2160

gagaccgtgg cccggcccct cttcctggag ttccccaagg actctagcac ctggactgtg 2220

gaccaccagc tcctgtgggg ggaggccctg ctcatcaccc cagtgctcca ggccgggaag 2280

gccgaagtga ctggctactt ccccttgggc acatggtacg acctgcagac ggtgccagta 2340

gaggcccttg gcagcctccc acccccacct gcagctcccc gtgagccagc catccacagc 2400

gaggggcagt gggtgacgct gccggccccc ctggacacca tcaacgtcca cctccgggct 2460

gggtacatca tccccctgca gggccctggc ctcacaacca cagagtcccg ccagcagccc 2520

atggccctgg ctgtggccct gaccaagggt ggggaggccc gaggggagct gttctgggac 2580

gatggagaga gcctggaagt gctggagcga ggggcctaca cacaggtcat cttcctggcc 2640

aggaataaca cgatcgtgaa tgagctggta cgtgtgacca gtgagggagc tggcctgcag 2700

ctgcagaagg tgactgtcct gggcgtggcc acggcgcccc agcaggtcct ctccaacggt 2760

gtccctgtct ccaacttcac ctacagcccc gacaccaagg tcctggacat ctgtgtctcg 2820

ctgttgatgg gagagcagtt tctcgtcagc tggtgttag 2859

<210> 9

<211> 2778

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAwt w/o sp

<400> 9

gggcacatcc tactccatga tttcctgctg gttccccgag agctgagtgg ctcctcccca 60

gtcctggagg agactcaccc agctcaccag cagggagcca gcagaccagg gccccggggat 120

gcccaggcac accccgggcg gccgcgagca gtgcccacac agtgcgacgt cccccccaac 180

agccgcttcg attgcgcccc tgacaaggcc atcacccagg aacagtgcga ggcccgcggc 240

tgttgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 300

ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatgggc 360

tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 420

cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 480

aacaggcgct acgaggtgcc cttggagacc ccgcatgtcc acagccgggc accgtcccca 540

ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcgccg gcagctggac 600

ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 660

ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 720

atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 780

ggtgcgaacc tctacgggtc tcaccctttc tacctggcgc tggaggacgg cgggtcggca 840

cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 900

cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctggg cccagagccc 960

aagagcgtgg tgcagcagta cctggacgtt gtgggatacc cgttcatgcc gccatactgg 1020

ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1080

gtggagaaca tgaccagggc ccacttcccc ctggacgtcc agtggaacga cctggactac 1140

atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1200

gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1260

agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1320

atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1380

cccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1440

gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcagg 1500

ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1560

gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1620

cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1680

gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 1740

cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 1800

gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 1860

ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 1920

taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 1980

agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2040

cacctctaca cactgttcca ccaggcccac gtcgcggggg agaccgtggc ccggcccctc 2100

ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2160

gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2220

cccttgggca catggtacga cctgcagacg gtgccagtag aggcccttgg cagcctccca 2280

cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2340

ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2400

ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2460

accaagggtg gggaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2520

ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2580

gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2640

ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 2700

tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 2760

ctcgtcagct ggtgttag 2778

<210> 10

<211> 2778

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco1 w/o sp

<400> 10

ggccatatcc tgctgcacga ctttctacta gtgcccagag agctgagcgg cagctctccc 60

gtgctggaag aaacacaccc tgcccatcag cagggcgcct ctagacctgg acctagagat 120

gcccaggccc accccggcag acctagagct gtgcctaccc agtgtgacgt gccccccaac 180

agcagattcg actgcgcccc tgacaaggcc atcacccagg aacagtgcga ggccagaggc 240

tgctgctaca tccctgccaa gcagggactg cagggcgctc agatgggaca gccctggtgc 300

ttcttcccac cctcctaccc cagctacaag ctggaaaacc tgagcagcag cgagatgggc 360

tacaccgcca ccctgaccag aaccaccccc acattcttcc caaaggacat cctgaccctg 420

cggctggacg tgatgatgga aaccgagaac cggctgcact tcaccatcaa ggaccccgcc 480

aatcggagat acgaggtgcc cctggaaacc ccccacgtgc actctagagc ccccagccct 540

ctgtacagcg tggaattcag cgaggaaccc ttcggcgtga tcgtgcggag acagctggat 600

ggcagagtgc tgctgaacac caccgtggcc cctctgttct tcgccgacca gttcctgcag 660

ctgagcacca gcctgcccag ccagtacatc acaggactgg ccgagcacct gagccccctg 720

atgctgagca catcctggac ccggatcacc ctgtggaaca gggatctggc ccctacccct 780

ggcgccaatc tgtacggcag ccaccctttc tacctggccc tggaagatgg cggatctgcc 840

cacggagtgt ttctgctgaa ctccaacgcc atggacgtgg tgctgcagcc tagccctgcc 900

ctgtcttgga gaagcacagg cggcatcctg gatgtgtaca tctttctggg ccccgagccc 960

aagagcgtgg tgcagcagta tctggatgtc gtgggctacc ccttcatgcc cccttactgg 1020

ggcctggggat tccacctgtg cagatggggc tactccagca ccgccatcac cagacaggtg 1080

gtggaaaaca tgaccagagc ccacttccca ctggatgtgc agtggaacga cctggactac 1140

atggacagca gacgggactt caccttcaac aaggacggct tccgggactt ccccgccatg 1200

gtgcaggaac tgcatcaggg cggcagacgg tacatgatga tcgtggatcc cgccatcagc 1260

tcctctggcc ctgccggctc ttacagaccc tacgacgagg gcctgcggag aggcgtgttc 1320

atcaccaacg agacaggcca gcccctgatc ggcaaagtgt ggcctggcag cacagccttc 1380

cccgacttca ccaatcctac cgccctggct tggtgggagg acatggtggc cgagttccac 1440

gaccaggtgc ccttcgacgg catgtggatc gacatgaacg agcccagcaa cttcatccgg 1500

ggcagcgagg atggctgccc caacaacgaa ctggaaaatc ccccttacgt gcccggcgtc 1560

gtgggcggaa cactgcaggc cgctacaatc tgtgccagca gccaccagtt tctgagcacc 1620

cactacaacc tgcacaacct gtacggcctg accgaggcca ttgccagcca ccgcgctctc 1680

gtgaaagcca gaggcacacg gcccttcgtg atcagcagaa gcacctttgc cggccacggc 1740

agatacgccg gacattggac tggcgacgtg tggtcctctt gggagcagct ggcctctagc 1800

gtgcccgaga tcctgcagtt caatctgctg ggcgtgccac tcgtgggcgc cgatgtgtgt 1860

ggcttcctgg gcaacacctc cgaggaactg tgtgtgcggt ggacacagct gggcgccttc 1920

taccctttca tgagaaacca caacagcctg ctgagcctgc cccaggaacc ctacagcttt 1980

agcgagcctg cacagcaggc catgcggaag gccctgacac tgagatacgc tctgctgccc 2040

cacctgtaca ccctgtttca ccaggcccat gtggccggcg agacagtggc cagacctctg 2100

tttctggaat tccccaagga cagcagcacc tggaccgtgg accatcagct gctgtgggga 2160

gaggctctgc tgattacccc agtgctgcag gcaggcaagg ccgaagtgac cggctacttt 2220

cccctgggca cttggtacga cctgcagacc gtgcctgtgg aagccctggg atctctgcct 2280

ccacctcctg ccgctcctag agagcctgcc attcactctg agggccagtg ggtcacactg 2340

cctgcccccc tggataccat caacgtgcac ctgagggccg gctacatcat accactgcag 2400

ggacctggcc tgaccaccac cgagtctaga cagcagccaa tggccctggc cgtggccctg 2460

accaaaggcg gagaagctag gggcgagctg ttctgggacg atggcgagag cctggaagtg 2520

ctggaaagag gcgcctatac ccaagtgatc ttcctggccc ggaacaacac catcgtgaac 2580

gagctggtgc gcgtgacctc tgaaggcgct ggactgcagc tgcagaaagt gaccgtgctg 2640

ggagtggcca cagcccctca gcaggtgctg tctaatggcg tgcccgtgtc caacttcacc 2700

tacagccccg acaccaaggt gctggacatc tgcgtgtcac tgctgatggg agagcagttt 2760

ctggtgtcct ggtgctga 2778

<210> 11

<211> 2778

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco2 w/o sp

<400> 11

ggacacatcc tgctgcacga cttcctgttg gtgcctagag agctgagcgg atcatcccca 60

gtgctggagg agactcatcc tgctcaccaa cagggagctt ccagaccagg accgagagac 120

gcccaagccc atcctggtag accaagagct gtgcctaccc aatgcgacgt gccacccaac 180

tcccgattcg actgcgcgcc agataaggct attacccaag agcagtgtga agccagaggt 240

tgctgctaca tcccagcgaa gcaaggattg caaggcgccc aaatgggaca accttggtgt 300

ttcttccccc cttcgtaccc atcatataaa ctcgaaaacc tgtcctcttc ggaaatgggt 360

tatactgcca ccctcaccag aactactcct actttcttcc cgaaagacat cttgaccttg 420

aggctggacg tgatgatgga gactgaaaac cggctgcatt tcactatcaa agatcctgcc 480

aatcggcgat acgaggtccc tctggaaacc cctcacgtgc actcacgggc tccttctccg 540

ctttactccg tcgaattctc tgaggaaccc ttcggagtga tcgttagacg ccagctggat 600

ggtagagtgc tgttgaacac tactgtggcc ccacttttct tcgctgacca gtttctgcaa 660

ctgtccactt ccctgccatc ccagtacatt actggactcg ccgaacacct gtcgccactg 720

atgctctcga cctcttggac tagaatcact ttgtggaaca gagacttggc ccctactccg 780

ggagcaaatc tgtacggaag ccaccctttt tacctggcgc tcgaagatgg cggatccgct 840

cacggagtgt tcctgctgaa tagcaacgca atggacgtgg tgctgcaacc ttcccctgca 900

ctcagttgga gaagtaccgg gggtattctg gacgtgtaca tcttcctcgg accagaaccc 960

aagagcgtgg tgcagcaata tctggacgtg gtcggatacc cttttatgcc tccttatactgg 1020

ggactggggat tccacctttg ccgttggggc tactcatcca ccgccattac cagacaggtg 1080

gtggagaata tgaccagagc ccacttccct ctcgacgtgc agtggaacga tctggactat 1140

atggactccc ggagagattt caccttcaac aaggacgggt tccgcgattt tcccgcgatg 1200

gttcaagagc tccaccagggg tggtcgaaga tatatgatga tcgtcgaccc agccatttcg 1260

agcagcggac ccgctggatc ttatagacct tacgacgaag gccttaggag aggagtgttc 1320

atcacaaacg agactggaca gcctttgatc ggtaaagtgt ggcctggatc aaccgccttt 1380

cctgacttta ccaatcccac tgccttggct tggtgggagg acatggtggc cgaattccac 1440

gaccaagtcc cctttgatgg aatgtggatc gatatgaacg aaccaagcaa ttttatcaga 1500

ggttccgaag acggttgccc caacaacgaa ctggaaaacc ctccttatgt gcccggagtc 1560

gtgggcggaa cattacaggc cgcgactatt tgcgccagca gccaccaatt cctgtccact 1620

cactacaacc tccacaacct ttatggatta accgaagcta ttgcaagtca cagggctctg 1680

gtgaaggcta gagggactag gccctttgtg atctcccgat ccacctttgc cggacacggg 1740

agatacgccg gtcactggac tggtgacgtg tggagctcat gggaacaact ggcctcctcc 1800

gtgccggaaa tcttacagtt caaccttctg ggtgtccctc ttgtcggagc agacgtgtgt 1860

gggtttcttg gtaacacctc cgaggaactg tgtgtgcgct ggactcaact gggtgcattc 1920

tacccattca tgagaaacca caactccttg ctgtccctgc cacaagagcc ctactcgttc 1980

agcgagcctg cacaacaggc tatgcggaag gcactgaccc tgagatacgc cctgcttcca 2040

cacttataca ctctcttcca tcaagcgcat gtggcaggag aaaccgttgc aaggcctctt 2100

ttccttgaat tccccaagga ttcctcgact tggacggtgg atcatcagct gctgtgggga 2160

gaagctctgc tgattactcc agtgttgcaa gccggaaaag ctgaggtgac cggatacttt 2220

ccgctgggaa cctggtacga cctccagact gtccctgttg aagcccttgg atcactgcct 2280

ccgcctccgg cagctccacg cgaaccagct atacattccg agggacagtg ggttacatta 2340

ccagctcctc tggacacaat caacgtccac ttaagagctg gctacattat ccctctgcaa 2400

ggaccaggac tgactacgac cgagagcaga cagcagccaa tggcactggc tgtggctctg 2460

accaagggag gggaagctag aggagaactc ttctgggatg atggggagtc ccttgaagtg 2520

ctggaaagag gcgcttacac tcaagtcatt ttccttgcac ggaacaacac cattgtgaac 2580

gaattggtgc gagtgaccag cgaaggagct ggacttcaac tgcagaaggt cactgtgctc 2640

ggagtggcta ccgctcctca gcaagtgctg tcgaatggag tccccgtgtc aaactttacc 2700

tactcccctg acactaaggt gctcgacatt tgcgtgtccc tcctgatggg agagcagttc 2760

cttgtgtcctggtgttga 2778

<210> 12

<211> 2754

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco1-Δ-8 w/o sp

<400> 12

ctactagtgc ccagagagct gagcggcagc tctcccgtgc tggaagaaac acaccctgcc 60

catcagcagg gcgcctctag acctggacct agagatgccc aggcccaccc cggcagacct 120

agagctgtgc ctacccagtg tgacgtgccc cccaacagca gattcgactg cgcccctgac 180

aaggccatca cccaggaaca gtgcgaggcc agaggctgct gctacatccc tgccaagcag 240

ggactgcagg gcgctcagat gggacagccc tggtgcttct tcccaccctc ctaccccagc 300

tacaagctgg aaaacctgag cagcagcgag atgggctaca ccgccaccct gaccagaacc 360

acccccacat tcttcccaaa ggacatcctg accctgcggc tggacgtgat gatggaaacc 420

gagaaccggc tgcacttcac catcaaggac cccgccaatc ggagatacga ggtgcccctg 480

gaaacccccc acgtgcactc tagagccccc agccctctgt acagcgtgga attcagcgag 540

gaacccttcg gcgtgatcgt gcggagacag ctggatggca gagtgctgct gaacaccacc 600

gtggcccctc tgttcttcgc cgaccagttc ctgcagctga gcaccagcct gcccagccag 660

tacatcacag gactggccga gcacctgagc cccctgatgc tgagcacatc ctggacccgg 720

atcaccctgt ggaacaggga tctggcccct acccctggcg ccaatctgta cggcagccac 780

cctttctacc tggccctgga agatggcgga tctgcccacg gagtgtttct gctgaactcc 840

aacgccatgg acgtggtgct gcagcctagc cctgccctgt cttggagaag cacaggcggc 900

atcctggatg tgtacatctt tctgggcccc gagcccaaga gcgtggtgca gcagtatctg 960

gatgtcgtgg gctacccctt catgccccct tactggggcc tgggattcca cctgtgcaga 1020

tggggctact ccagcaccgc catcaccaga caggtggtgg aaaacatgac cagagcccac 1080

ttcccactgg atgtgcagtg gaacgacctg gactacatgg acagcagacg ggacttcacc 1140

ttcaacaagg acggcttccg ggacttcccc gccatggtgc aggaactgca tcagggcggc 1200

agacggtaca tgatgatcgt ggatcccgcc atcagctcct ctggccctgc cggctcttac 1260

agaccctacg acgagggcct gcggagaggc gtgttcatca ccaacgagac aggccagccc 1320

ctgatcggca aagtgtggcc tggcagcaca gccttccccg acttcaccaa tcctaccgcc 1380

ctggcttggt gggaggacat ggtggccgag ttccacgacc aggtgccctt cgacggcatg 1440

tggatcgaca tgaacgagcc cagcaacttc atccggggca gcgaggatgg ctgccccaac 1500

aacgaactgg aaaatccccc ttacgtgccc ggcgtcgtgg gcggaacact gcaggccgct 1560

acaatctgtg ccagcagcca ccagtttctg agcacccact acaacctgca caacctgtac 1620

ggcctgaccg aggccattgc cagccaccgc gctctcgtga aagccagagg cacacggccc 1680

ttcgtgatca gcagaagcac ctttgccggc cacggcagat acgccggaca ttggactggc 1740

gacgtgtggt cctcttggga gcagctggcc tctagcgtgc ccgagatcct gcagttcaat 1800

ctgctgggcg tgccactcgt gggcgccgat gtgtgtggct tcctgggcaa cacctccgag 1860

gaactgtgtg tgcggtggac acagctgggc gccttctacc ctttcatgag aaaccacaac 1920

agcctgctga gcctgcccca ggaaccctac agctttagcg agcctgcaca gcaggccatg 1980

cggaaggccc tgacactgag atacgctctg ctgccccacc tgtacaccct gtttcaccag 2040

gcccatgtgg ccggcgagac agtggccaga cctctgtttc tggaattccc caaggacagc 2100

agcacctgga ccgtggacca tcagctgctg tggggagagg ctctgctgat taccccagtg 2160

ctgcaggcag gcaaggccga agtgaccggc tactttcccc tgggcacttg gtacgacctg 2220

cagaccgtgc ctgtggaagc cctgggatct ctgcctccac ctcctgccgc tcctagagag 2280

cctgccattc actctgaggg ccagtgggtc acactgcctg cccccctgga taccatcaac 2340

gtgcacctga gggccggcta catcatacca ctgcagggac ctggcctgac caccacccgag 2400

tctagacagc agccaatggc cctggccgtg gccctgacca aaggcggaga agctaggggc 2460

gagctgttct gggacgatgg cgagagcctg gaagtgctgg aaagaggcgc ctatacccaa 2520

gtgatcttcc tggccccggaa caacaccatc gtgaacgagc tggtgcgcgt gacctctgaa 2580

ggcgctggac tgcagctgca gaaagtgacc gtgctgggag tggccacagc ccctcagcag 2640

gtgctgtcta atggcgtgcc cgtgtccaac ttcacctaca gccccgacac caaggtgctg 2700

gacatctgcg tgtcactgct gatggggagag cagtttctgg tgtcctggtg ctga 2754

<210> 13

<211> 2754

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco2-Δ8 w/o sp

<400> 13

ctgttggtgc ctagagagct gagcggatca tccccagtgc tggaggagac tcatcctgct 60

caccaacagg gagcttccag accaggaccg agagacgccc aagcccatcc tggtagacca 120

agagctgtgc ctacccaatg cgacgtgcca cccaactccc gattcgactg cgcgccagat 180

aaggctatta cccaagagca gtgtgaagcc agaggttgct gctacatccc agcgaagcaa 240

ggattgcaag gcgcccaaat gggacaacct tggtgtttct tccccccttc gtacccatca 300

tataaactcg aaaacctgtc ctcttcggaa atgggttata ctgccaccct caccagaact 360

actcctactt tcttcccgaa agacatcttg accttgaggc tggacgtgat gatggagact 420

gaaaaccggc tgcatttcac tatcaaagat cctgccaatc ggcgatacga ggtccctctg 480

gaaacccctc acgtgcactc acgggctcct tctccgcttt actccgtcga attctctgag 540

gaacccttcg gagtgatcgt tagacgccag ctggatggta gagtgctgtt gaacactact 600

gtggccccac ttttcttcgc tgaccagttt ctgcaactgt ccacttccct gccatcccag 660

tacattactg gactcgccga acacctgtcg ccactgatgc tctcgacctc ttggactaga 720

atcactttgt ggaacagaga cttggcccct actccgggag caaatctgta cggaagccac 780

cctttttacc tggcgctcga agatggcgga tccgctcacg gagtgttcct gctgaatagc 840

aacgcaatgg acgtggtgct gcaaccttcc cctgcactca gttggagaag taccgggggt 900

attctggacg tgtacatctt cctcggacca gaacccaaga gcgtggtgca gcaatatctg 960

gacgtggtcg gatacccttt tatgcctcct tactggggac tgggattcca cctttgccgt 1020

tggggctact catccaccgc cattaccaga caggtggtgg agaatatgac cagagcccac 1080

ttccctctcg acgtgcagtg gaacgatctg gactatatgg actcccggag agatttcacc 1140

ttcaacaagg acgggttccg cgattttccc gcgatggttc aagagctcca ccagggtggt 1200

cgaagatata tgatgatcgt cgacccagcc atttcgagca gcggacccgc tggatctttat 1260

agaccttacg acgaaggcct taggagagga gtgttcatca caaacgagac tggacagcct 1320

ttgatcggta aagtgtggcc tggatcaacc gcctttcctg actttaccaa tcccactgcc 1380

ttggcttggt gggaggacat ggtggccgaa ttccacgacc aagtcccctt tgatggaatg 1440

tggatcgata tgaacgaacc aagcaatttt atcagaggtt ccgaagacgg ttgccccaac 1500

aacgaactgg aaaaccctcc ttatgtgccc ggagtcgtgg gcggaacatt acaggccgcg 1560

actatttgcg ccagcagcca ccaattcctg tccactcact acaacctcca caacctttat 1620

ggattaaccg aagctattgc aagtcacagg gctctggtga aggctagagg gactaggccc 1680

tttgtgatct cccgatccac ctttgccgga cacggggagat acgccggtca ctggactggt 1740

gacgtgtgga gctcatggga acaactggcc tcctccgtgc cggaaatctt acagttcaac 1800

cttctgggtg tccctcttgt cggagcagac gtgtgtgggt ttcttggtaa cacctccgag 1860

gaactgtgtg tgcgctggac tcaactgggt gcattctacc cattcatgag aaaccacaac 1920

tccttgctgt ccctgccaca agagccctac tcgttcagcg agcctgcaca acaggctatg 1980

cggaaggcac tgaccctgag atacgccctg cttccacact tatacactct cttccatcaa 2040

gcgcatgtgg caggagaaac cgttgcaagg cctcttttcc ttgaattccc caaggattcc 2100

tcgacttgga cggtggatca tcagctgctg tggggagaag ctctgctgat tactccagtg 2160

ttgcaagccg gaaaagctga ggtgaccgga tactttccgc tgggaacctg gtacgacctc 2220

cagactgtcc ctgttgaagc ccttggatca ctgcctccgc ctccggcagc tccacgcgaa 2280

ccagctatac attccgaggg acagtgggtt acattaccag ctcctctgga cacaatcaac 2340

gtccacttaa gagctggcta cattatccct ctgcaaggac caggactgac tacgaccgag 2400

agcagacagc agccaatggc actggctgtg gctctgacca agggagggga agctagagga 2460

gaactcttct gggatgatgg ggagtccctt gaagtgctgg aaagaggcgc ttacactcaa 2520

gtcattttcc ttgcacggaa caacaccatt gtgaacgaat tggtgcgagt gaccagcgaa 2580

ggagctggac ttcaactgca gaaggtcact gtgctcggag tggctaccgc tcctcagcaa 2640

gtgctgtcga atggagtccc cgtgtcaaac tttacctact cccctgacac taaggtgctc 2700

gacatttgcg tgtccctcct gatgggag cagttccttg tgtcctggtg ttga 2754

<210> 14

<211> 397

<212> DNA

<213> Artificial sequence

<220>

<223> hAAT promoter

<400> 14

gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 60

agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 120

gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 180

ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 240

tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 300

cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 360

cagcttcagg caccacccact gacctgggac agtgaat 397

<210> 15

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> ApoE control area

<400> 15

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg g 321

<210> 16

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> HBB2 intron

<400> 16

gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60

cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120

gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180

atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240

aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300

ttattttatg gttggggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360

tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg gtctgtgtgc 420

tggcccatca ctttggcaaaa g 441

<210> 17

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> Modified HBB2 intron

<400> 17

gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60

cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120

gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180

atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240

aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300

ttattttctg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360

tcttgttcat acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc 420

tggcccatca ctttggcaaaa g 441

<210> 18

<211> 1438

<212> DNA

<213> Artificial sequence

<220>

<223> FIX intron

<400> 18

ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 60

gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120

acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180

atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 240

tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300

aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 360

tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 420

ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480

aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540

cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600

ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 660

tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720

agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 780

tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 840

aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900

accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960

cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020

tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080

agggtgatgg atcactttgc aaagatcctc aatgagctat tttcaagtga tgacaaagtg 1140

tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200

agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260

cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320

aaagagaaat tggctttcag attatttgga ttaaaaacaa agactttctt aagagatgta 1380

aaattttcat gatgttttct tttttgctaa aactaaagaa ttatctttt acatttca 1438

<210> 19

<211> 1438

<212> DNA

<213> Artificial sequence

<220>

<223> Modified FIX intron

<400> 19

ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 60

gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120

acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180

atttttaaaa ctaaatagat cgacattgct tttgttgcat ttatgtttaa taaacactgt 240

tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300

aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 360

tttttgtttg gacttaccac tttgaaatca aattgggaaa caaaagcaca aacaatggcc 420

ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480

aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540

cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600

ggaaaaaatc attttgtctc tttgtcaaac atcttggagt tgatatttgg ggaaacacaa 660

tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720

agttagctat tgcaacatat atcactttgt tttttcacaa ctacagtgac tttttgtatt 780

tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc ttgttctcac 840

aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900

accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960

cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020

tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080

agggtgttgg atcactttgc aaagatcctc attgagctat tttcaagtgt tgacaaagtg 1140

tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200

agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260

cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320

aaagagaaat tggctttcag attatttgga ttaaaaacaa agactttctt aagagatgta 1380

aaattttctt gttgttttct tttttgctaa aactaaagaa ttatctttt acatttca 1438

<210> 20

<211> 881

<212> DNA

<213> Artificial sequence

<220>

<223> Chicken beta-globin intron

<400> 20

gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60

gccccggctc tgactgaccg cgttatccc acaggtgagc gggcgggacg gcccttctcc 120

tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 180

aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240

cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300

ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360

gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420

gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480

cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540

tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600

ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660

cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 720

gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggggc gccgccgcac 780

cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 840

gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881

<210> 21

<211> 881

<212> DNA

<213> Artificial sequence

<220>

<223> Modified chicken β-globin intron

<400> 21

gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60

gccccggctc tgactgaccg cgttatccc acaggtgagc gggcgggacg gcccttctcc 120

tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 180

aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240

cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300

ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360

gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420

gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480

cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540

tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600

ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660

cggctgtcga ggcgcggcga gccgcagcca ttgccttttt tggtaatcgt gcgagagggc 720

gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggggc gccgccgcac 780

cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaat tgggcgggga 840

gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881

<210> 22

<211> 4318

<212> DNA

<213> Artificial sequence

<220>

<223> Construct: sp2+hGAAwt-Δ-8

<400> 22

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccacccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

agaggtttca tattgctaat agcagctaca atccagctac cattctgctt ttattttctg 1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctctttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgccg tcttctgtct cgtggggcat cctcctgctg 1320

gcaggcctgt gctgcctggt ccctgtctcc ctggctctgc tggttccccg agagctgagt 1380

ggctcctccc cagtcctgga ggagactcac ccagctcacc agcagggagc cagcagacca 1440

gggccccggg atgcccaggc acaccccggg cggccgcgag cagtgcccac acagtgcgac 1500

gtccccccca acagccgctt cgattgcgcc cctgacaagg ccatcaccca ggaacagtgc 1560

gaggcccgcg gctgttgcta catccctgca aagcaggggc tgcagggagc ccagatgggg 1620

cagccctggt gcttcttccc acccagctac cccagctaca agctggagaa cctgagctcc 1680

tctgaaatgg gctacacggc caccctgacc cgtaccaccc ccaccttctt ccccaaggac 1740

atcctgaccc tgcggctgga cgtgatgatg gagactgaga accgcctcca cttcacgatc 1800

aaagatccag ctaacaggcg ctacgaggtg cccttggaga ccccgcatgt ccacagccgg 1860

gcaccgtccc cactctacag cgtggagttc tccgaggagc ccttcggggt gatcgtgcgc 1920

cggcagctgg acggccgcgt gctgctgaac acgacggtgg cgcccctgtt ctttgcggac 1980

cagttccttc agctgtccac ctcgctgccc tcgcagtata tcacaggcct cgccgagcac 2040

ctcagtcccc tgatgctcag caccagctgg accaggatca ccctgtggaa ccgggacctt 2100

gcgcccacgc ccggtgcgaa cctctacggg tctcaccctt tctacctggc gctggaggac 2160

ggcgggtcgg cacacggggt gttcctgcta aacagcaatg ccatggatgt ggtcctgcag 2220

ccgagccctg cccttagctg gaggtcgaca ggtggggatcc tggatgtcta catcttcctg 2280

ggcccagc ccaagagcgt ggtgcagcag tacctggacg ttgtgggata cccgttcatg 2340

ccgccatact ggggcctggg cttccacctg tgccgctggg gctactcctc caccgctatc 2400

acccgccagg tggtggagaa catgaccagg gcccacttcc ccctggacgt ccagtggaac 2460

gacctggact acatggactc ccggagggac ttcacgttca acaaggatgg cttccgggac 2520

ttcccggcca tggtgcagga gctgcaccag ggcggccggc gctacatgat gatcgtggat 2580

cctgccatca gcagctcggg ccctgccggg agctacaggc cctacgacga gggtctgcgg 2640

aggggggttt tcatcaccaa cgagaccggc cagccgctga ttgggaaggt atggcccggg 2700

tccactgcct tccccgactt caccaacccc acagccctgg cctggtggga ggacatggtg 2760

gctgagttcc atgaccaggt gcccttcgac ggcatgtgga ttgacatgaa cgagccttcc 2820

aacttcatca ggggctctga ggacggctgc cccaacaatg agctggagaa cccaccctac 2880

gtgcctgggg tggttggggg gaccctccag gcggccacca tctgtgcctc cagccaccag 2940

tttctctcca cacactacaa cctgcacaac ctctacggcc tgaccgaagc catcgcctcc 3000

cacagggcgc tggtgaaggc tcgggggaca cgcccatttg tgatctcccg ctcgaccttt 3060

gctggccacg gccgatacgc cggccactgg acgggggacg tgtggagctc ctgggagcag 3120

ctcgcctcct ccgtgccaga aatcctgcag tttaacctgc tgggggtgcc tctggtcggg 3180

gccgacgtct gcggcttcct gggcaacacc tcagaggagc tgtgtgtgcg ctggacccag 3240

ctgggggcct tctacccctt catgcggaac cacaacagcc tgctcagtct gccccaggag 3300

ccgtacagct tcagcgagcc ggcccagcag gccatgagga aggccctcac cctgcgctac 3360

gcactcctcc cccacctcta cacactgttc caccaggccc acgtcgcggg ggagaccgtg 3420

gcccggcccc tcttcctgga gttccccaag gactctagca cctggactgt ggaccaccag 3480

ctcctgtggg gggaggccct gctcatcacc ccagtgctcc aggccgggaa ggccgaagtg 3540

actggctact tccccttggg cacatggtac gacctgcaga cggtgccagt agaggccctt 3600

ggcagcctcc cacccccacc tgcagctccc cgtgagccag ccatccacag cgaggggcag 3660

tgggtgacgc tgccggcccc cctggacacc atcaacgtcc acctccgggc tgggtacatc 3720

atccccctgc aggggccctgg cctcacaacc acagagtccc gccagcagcc catggccctg 3780

gctgtggccc tgaccaaggg tggggaggcc cgaggggagc tgttctggga cgatggagag 3840

agcctggaag tgctggagcg aggggcctac acacaggtca tcttcctggc caggaataac 3900

acgatcgtga atgagctggt acgtgtgacc agtgaggggag ctggcctgca gctgcagaag 3960

gtgactgtcc tgggcgtggc cacggcgccc cagcaggtcc tctccaacgg tgtccctgtc 4020

tccaacttca cctacagccc cgacaccaag gtcctggaca tctgtgtctc gctgttgatg 4080

ggagagcagt ttctcgtcag ctggtgttag ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 23

<211> 4318

<212> DNA

<213> Artificial sequence

<220>

<223> Construct: sp2+hGAAco1-Δ-8

<400> 23

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccacccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

agaggtttca tattgctaat agcagctaca atccagctac cattctgctt ttattttctg 1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctctttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgcct agctctgtgt cctggggcat tctgctgctg 1320

gccggcctgt gttgtctggt gcctgtgtct ctggccctac tagtgcccag agagctgagc 1380

ggcagctctc ccgtgctgga agaaacacac cctgcccatc agcagggcgc ctctagacct 1440

ggacctagag atgcccaggc ccaccccggc agacctagag ctgtgcctac ccagtgtgac 1500

gtgcccccca acagcagatt cgactgcgcc cctgacaagg ccatcaccca ggaacagtgc 1560

gaggccagag gctgctgcta catccctgcc aagcagggac tgcagggcgc tcagatggga 1620

cagccctggt gcttcttccc accctcctac cccagctaca agctggaaaa cctgagcagc 1680

agcgagatgg gctacaccgc caccctgacc agaaccaccc ccacattctt cccaaaggac 1740

atcctgaccc tgcggctgga cgtgatgatg gaaaccgaga accggctgca cttcaccatc 1800

aaggaccccg ccaatcggag atacgaggtg cccctggaaa ccccccacgt gcactctaga 1860

gcccccagcc ctctgtacag cgtggaattc agcgaggaac ccttcggcgt gatcgtgcgg 1920

agacagctgg atggcagagt gctgctgaac accaccgtgg cccctctgtt cttcgccgac 1980

cagttcctgc agctgagcac cagcctgccc agccagtaca tcacaggact ggccgagcac 2040

ctgagccccc tgatgctgag cacatcctgg acccggatca ccctgtggaa cagggatctg 2100

gcccctaccc ctggcgccaa tctgtacggc agccaccctt tctacctggc cctggaagat 2160

ggcggatctg cccacggagt gtttctgctg aactccaacg ccatggacgt ggtgctgcag 2220

cctagccctg ccctgtcttg gagaagcaca ggcggcatcc tggatgtgta catctttctg 2280

ggccccgagc ccaagagcgt ggtgcagcag tatctggatg tcgtgggcta ccccttcatg 2340

cccccttact ggggcctggg attccacctg tgcagatggg gctactccag caccgccatc 2400

accagacagg tggtggaaaa catgaccaga gcccacttcc cactggatgt gcagtggaac 2460

gacctggact acatggacag cagacgggac ttcaccttca acaaggacgg cttccgggac 2520

ttccccgcca tggtgcagga actgcatcag ggcggcagac ggtacatgat gatcgtggat 2580

cccgccatca gctcctctgg ccctgccggc tcttacagac cctacgacga gggcctgcgg 2640

agaggcgtgt tcatcaccaa cgagacaggc cagcccctga tcggcaaagt gtggcctggc 2700

agcacagcct tccccgactt caccaatcct accgccctgg cttggtggga ggacatggtg 2760

gccgagttcc acgaccaggt gcccttcgac ggcatgtgga tcgacatgaa cgagcccagc 2820

aacttcatcc ggggcagcga ggatggctgc cccaacaacg aactggaaaa tcccccttac 2880

gtgcccggcg tcgtgggcgg aacactgcag gccgctacaa tctgtgccag cagccaccag 2940

tttctgagca cccactacaa cctgcacaac ctgtacggcc tgaccgaggc cattgccagc 3000

caccgcgctc tcgtgaaagc cagaggcaca cggcccttcg tgatcagcag aagcaccttt 3060

gccggccacg gcagatacgc cggacattgg actggcgacg tgtggtcctc ttggggagcag 3120

ctggcctcta gcgtgcccga gatcctgcag ttcaatctgc tgggcgtgcc actcgtgggc 3180

gccgatgtgt gtggcttcct gggcaacacc tccgaggaac tgtgtgtgcg gtggacacag 3240

ctgggcgcct tctacccttt catgagaaac cacaacagcc tgctgagcct gccccaggaa 3300

ccctacagct ttagcgagcc tgcacagcag gccatgcgga aggccctgac actgagatac 3360

gctctgctgc cccacctgta caccctgttt caccaggccc atgtggccgg cgagacagtg 3420

gccagacctc tgtttctgga attccccaag gacagcagca cctggaccgt ggaccatcag 3480

ctgctgtggg gagaggctct gctgattacc ccagtgctgc aggcaggcaa ggccgaagtg 3540

accggctact ttcccctggg cacttggtac gacctgcaga ccgtgcctgt ggaagccctg 3600

ggatctctgc ctccacctcc tgccgctcct agagagcctg ccattcactc tgagggccag 3660

tgggtcacac tgcctgcccc cctggatacc atcaacgtgc acctgagggc cggctacatc 3720

ataccactgc agggacctgg cctgaccacc accgagtcta gacagcagcc aatggccctg 3780

gccgtggccc tgaccaaagg cggagaagct aggggcgagc tgttctggga cgatggcgag 3840

agcctggaag tgctggaaag aggcgcctat acccaagtga tcttcctggc ccggaacaac 3900

accatcgtga acgagctggt gcgcgtgacc tctgaaggcg ctggactgca gctgcagaaa 3960

gtgaccgtgc tgggagtggc cacagcccct cagcaggtgc tgtctaatgg cgtgcccgtg 4020

tccaacttca cctacagccc cgacaccaag gtgctggaca tctgcgtgtc actgctgatg 4080

ggagagcagt ttctggtgtc ctggtgctga ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 24

<211> 4318

<212> DNA

<213> Artificial sequence

<220>

<223> Construct: sp2+hGAAco2-Δ-8

<400> 24

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccacccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

agaggtttca tattgctaat agcagctaca atccagctac cattctgctt ttattttctg 1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctctttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgcca tcgtcagtgt cttggggcat tcttctgctc 1320

gccggattgt gttgcctggt gcctgtctca ttggccctgt tggtgcctag agagctgagc 1380

ggatcatccc cagtgctgga ggagactcat cctgctcacc aacagggagc ttccagacca 1440

ggaccgagag acgcccaagc ccatcctggt agaccaagag ctgtgcctac ccaatgcgac 1500

gtgccaccca actcccgatt cgactgcgcg ccagataagg ctattaccca agagcagtgt 1560

gaagccagag gttgctgcta catcccagcg aagcaaggat tgcaaggcgc ccaaatggga 1620

caaccttggt gtttcttccc cccttcgtac ccatcatata aactcgaaaa cctgtcctct 1680

tcggaaatgg gttatactgc caccctcacc agaactactc ctactttctt cccgaaagac 1740

atcttgacct tgaggctgga cgtgatgatg gagactgaaa accggctgca tttcactatc 1800

aaagatcctg ccaatcggcg atacgaggtc cctctggaaa cccctcacgt gcactcacgg 1860

gctccttctc cgctttactc cgtcgaattc tctgaggaac ccttcggagt gatcgttaga 1920

cgccagctgg atggtagagt gctgttgaac actactgtgg ccccactttt cttcgctgac 1980

cagtttctgc aactgtccac ttccctgcca tcccagtaca ttactggact cgccgaacac 2040

ctgtcgccac tgatgctctc gacctcttgg actagaatca ctttgtggaa cagagacttg 2100

gcccctactc cgggagcaaa tctgtacgga agccaccctt tttacctggc gctcgaagat 2160

ggcggatccg ctcacggagt gttcctgctg aatagcaacg caatggacgt ggtgctgcaa 2220

ccttcccctg cactcagttg gagaagtacc gggggtattc tggacgtgta catcttcctc 2280

ggaccagaac ccaagagcgt ggtgcagcaa tatctggacg tggtcggata cccttttatg 2340

cctccttact ggggactggg attccacctt tgccgttggg gctactcatc caccgccatt 2400

accagacagg tggtggagaa tatgaccaga gcccacttcc ctctcgacgt gcagtggaac 2460

gatctggact atatggactc ccggagagat ttcaccttca acaaggacgg gttccgcgat 2520

tttcccgcga tggttcaaga gctccaccag ggtggtcgaa gatatatgat gatcgtcgac 2580

ccagccattt cgagcagcgg acccgctgga tcttatagac cttacgacga aggccttagg 2640

agaggagtgt tcatcacaaa cgagactgga cagcctttga tcggtaaagt gtggcctgga 2700

tcaaccgcct ttcctgactt taccaatccc actgccttgg cttggtggga ggacatggtg 2760

gccgaattcc acgaccaagt cccctttgat ggaatgtgga tcgatatgaa cgaaccaagc 2820

aattttatca gaggttccga agacggttgc cccaacaacg aactggaaaa ccctccttat 2880

gtgcccggag tcgtgggcgg aacattacag gccgcgacta tttgcgccag cagccaccaa 2940

ttcctgtcca ctcactacaa cctccacaac ctttatggat taaccgaagc tattgcaagt 3000

cacagggctc tggtgaaggc tagagggact aggccctttg tgatctcccg atccaccttt 3060

gccggacacg ggagatacgc cggtcactgg actggtgacg tgtggagctc atgggaacaa 3120

ctggcctcct ccgtgccgga aatcttacag ttcaaccttc tgggtgtccc tcttgtcgga 3180

gcagacgtgt gtgggtttct tggtaacacc tccgaggaac tgtgtgtgcg ctggactcaa 3240

ctgggtgcat tctacccatt catgagaaac cacaactcct tgctgtccct gccacaagag 3300

ccctactcgt tcagcgagcc tgcacaacag gctatgcgga aggcactgac cctgagatac 3360

gccctgcttc cacacttata cactctcttc catcaagcgc atgtggcagg agaaaccgtt 3420

gcaaggcctc ttttccttga attccccaag gattcctcga cttggacggt ggatcatcag 3480

ctgctgtggg gagaagctct gctgattact ccagtgttgc aagccggaaa agctgaggtg 3540

accggatact ttccgctggg aacctggtac gacctccaga ctgtccctgt tgaagccctt 3600

ggatcactgc ctccgcctcc ggcagctcca cgcgaaccag ctatacattc cgagggacag 3660

tgggttacat taccagctcc tctggacaca atcaacgtcc acttaagagc tggctacatt 3720

atccctctgc aaggaccagg actgactacg accgagagca gacagcagcc aatggcactg 3780

gctgtggctc tgaccaaggg aggggaagct agaggagaac tcttctggga tgatggggag 3840

tcccttgaag tgctggaaag aggcgcttac actcaagtca ttttccttgc acggaacaac 3900

accattgtga acgaattggt gcgagtgacc agcgaaggag ctggacttca actgcagaag 3960

gtcactgtgc tcggagtggc taccgctcct cagcaagtgc tgtcgaatgg agtccccgtg 4020

tcaaacttta cctactcccc tgacactaag gtgctcgaca tttgcgtgtc cctcctgatg 4080

ggagagcagt tccttgtgtc ctggtgttga ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 25

<211> 4300

<212> DNA

<213> Artificial sequence

<220>

<223> Construct: sp7+hGAAco1-Δ-8

<400> 25

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccacccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

agaggtttca tattgctaat agcagctaca atccagctac cattctgctt ttattttctg 1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctctttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatggcc tttctgtggc tgctgagctg ttgggccctg 1320

ctgggcacca ccttcggcct actagtgccc agagagctga gcggcagctc tcccgtgctg 1380

gaagaaacac accctgccca tcagcagggc gcctctagac ctggacctag agatgcccag 1440

gcccaccccg gcagacctag agctgtgcct acccagtgtg acgtgccccc caacagcaga 1500

ttcgactgcg cccctgacaa ggccatcacc caggaacagt gcgaggccag aggctgctgc 1560

tacatccctg ccaagcaggg actgcagggc gctcagatgg gacagccctg gtgcttcttc 1620

ccaccctcct accccagcta caagctggaa aacctgagca gcagcgagat gggctacacc 1680

gccaccctga ccagaaccac ccccacattc ttcccaaagg acatcctgac cctgcggctg 1740

gacgtgatga tggaaaccga gaaccggctg cacttcacca tcaaggaccc cgccaatcgg 1800

agatacgagg tgcccctgga aaccccccac gtgcactcta gagcccccag ccctctgtac 1860

agcgtggaat tcagcgagga acccttcggc gtgatcgtgc ggagacagct ggatggcaga 1920

gtgctgctga acaccaccgt ggcccctctg ttcttcgccg accagttcct gcagctgagc 1980

accagcctgc ccagccagta catcacagga ctggccgagc acctgagccc cctgatgctg 2040

agcacatcct ggacccggat caccctgtgg aacagggatc tggcccctac ccctggcgcc 2100

aatctgtacg gcagccaccc tttctacctg gccctggaag atggcggatc tgcccacgga 2160

gtgtttctgc tgaactccaa cgccatggac gtggtgctgc agcctagccc tgccctgtct 2220

tggagaagca caggcggcat cctggatgtg tacatctttc tgggccccga gcccaagagc 2280

gtggtgcagc agtatctgga tgtcgtgggc taccccttca tgcccccctta ctggggcctg 2340

ggattccacc tgtgcagatg gggctactcc agcaccgcca tcaccagaca ggtggtggaa 2400

aacatgacca gagcccactt cccactggat gtgcagtgga acgacctgga ctacatggac 2460

agcagacggg acttcacctt caacaaggac ggcttccggg acttccccgc catggtgcag 2520

gaactgcatc agggcggcag acggtacatg atgatcgtgg atcccgccat cagctcctct 2580

ggccctgccg gctcttacag accctacgac gagggcctgc ggagaggcgt gttcatcacc 2640

aacgagacag gccagcccct gatcggcaaa gtgtggcctg gcagcacagc cttccccgac 2700

ttcaccaatc ctaccgccct ggcttggtgg gaggacatgg tggccgagtt ccacgaccag 2760

gtgcccttcg acggcatgtg gatcgacatg aacgagccca gcaacttcat ccggggcagc 2820

gaggatggct gccccaacaa cgaactggaa aatccccctt acgtgcccgg cgtcgtgggc 2880

ggaacactgc aggccgctac aatctgtgcc agcagccacc agtttctgag cacccactac 2940

aacctgcaca acctgtacgg cctgaccgag gccattgcca gccaccgcgc tctcgtgaaa 3000

gccagaggca cacggccctt cgtgatcagc agaagcacct ttgccggcca cggcagatac 3060

gccggacatt ggactggcga cgtgtggtcc tcttggggagc agctggcctc tagcgtgccc 3120

gagatcctgc agttcaatct gctgggcgtg ccactcgtgg gcgccgatgt gtgtggcttc 3180

ctgggcaaca cctccgagga actgtgtgtg cggtggacac agctgggcgc cttctaccct 3240

ttcatgagaa accacaacag cctgctgagc ctgccccagg aaccctacag ctttagcgag 3300

cctgcacagc aggccatgcg gaaggccctg acactgagat acgctctgct gccccacctg 3360

tacaccctgt ttcaccaggc ccatgtggcc ggcgagacag tggccagacc tctgtttctg 3420

gaattcccca aggacagcag cacctggacc gtggaccatc agctgctgtg gggagaggct 3480

ctgctgatta ccccagtgct gcaggcaggc aaggccgaag tgaccggcta ctttcccctg 3540

ggcacttggt acgacctgca gaccgtgcct gtggaagccc tgggatctct gcctccacct 3600

cctgccgctc ctagagagcc tgccattcac tctgagggcc agtgggtcac actgcctgcc 3660

cccctggata ccatcaacgt gcacctgagg gccggctaca tcataccact gcagggacct 3720

ggcctgacca ccaccgagtc tagacagcag ccaatggccc tggccgtggc cctgaccaaa 3780

ggcggagaag ctaggggcga gctgttctgg gacgatggcg agagcctgga agtgctggaa 3840

agaggcgcct atacccaagt gatcttcctg gcccggaaca acaccatcgt gaacgagctg 3900

gtgcgcgtga cctctgaagg cgctggactg cagctgcaga aagtgaccgt gctggggagtg 3960

gccacagccc ctcagcaggt gctgtctaat ggcgtgcccg tgtccaactt cacctacagc 4020

cccgacacca aggtgctgga catctgcgtg tcactgctga tgggagagca gtttctggtg 4080

tcctggtgct gactcgagag atctaccggt gaattcaccg cgggtttaaa ctgtgccttc 4140

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4200

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4260

tcattctatt ctggggggtg gggtgggggc tagctctaga 4300

<210> 26

<211> 4198

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco1-Δ-42

<400> 26

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccacccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

agaggtttca tattgctaat agcagctaca atccagctac cattctgctt ttattttctg 1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctctttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatggcc tttctgtggc tgctgagctg ttgggccctg 1320

ctgggcacca ccttcggcgc ccaccccggc agacctagag ctgtgcctac ccagtgtgac 1380

gtgcccccca acagcagatt cgactgcgcc cctgacaagg ccatcaccca ggaacagtgc 1440

gaggccagag gctgctgcta catccctgcc aagcagggac tgcagggcgc tcagatggga 1500

cagccctggt gcttcttccc accctcctac cccagctaca agctggaaaa cctgagcagc 1560

agcgagatgg gctacaccgc caccctgacc agaaccaccc ccacattctt cccaaaggac 1620

atcctgaccc tgcggctgga cgtgatgatg gaaaccgaga accggctgca cttcaccatc 1680

aaggaccccg ccaatcggag atacgaggtg cccctggaaa ccccccacgt gcactctaga 1740

gcccccagcc ctctgtacag cgtggaattc agcgaggaac ccttcggcgt gatcgtgcgg 1800

agacagctgg atggcagagt gctgctgaac accaccgtgg cccctctgtt cttcgccgac 1860

cagttcctgc agctgagcac cagcctgccc agccagtaca tcacaggact ggccgagcac 1920

ctgagccccc tgatgctgag cacatcctgg acccggatca ccctgtggaa cagggatctg 1980

gcccctaccc ctggcgccaa tctgtacggc agccaccctt tctacctggc cctggaagat 2040

ggcggatctg cccacggagt gtttctgctg aactccaacg ccatggacgt ggtgctgcag 2100

cctagccctg ccctgtcttg gagaagcaca ggcggcatcc tggatgtgta catctttctg 2160

ggccccgagc ccaagagcgt ggtgcagcag tatctggatg tcgtgggcta ccccttcatg 2220

cccccttact ggggcctggg attccacctg tgcagatggg gctactccag caccgccatc 2280

accagacagg tggtggaaaa catgaccaga gcccacttcc cactggatgt gcagtggaac 2340

gacctggact acatggacag cagacgggac ttcaccttca acaaggacgg cttccgggac 2400

ttccccgcca tggtgcagga actgcatcag ggcggcagac ggtacatgat gatcgtggat 2460

cccgccatca gctcctctgg ccctgccggc tcttacagac cctacgacga gggcctgcgg 2520

agaggcgtgt tcatcaccaa cgagacaggc cagcccctga tcggcaaagt gtggcctggc 2580

agcacagcct tccccgactt caccaatcct accgccctgg cttggtggga ggacatggtg 2640

gccgagttcc acgaccaggt gcccttcgac ggcatgtgga tcgacatgaa cgagcccagc 2700

aacttcatcc ggggcagcga ggatggctgc cccaacaacg aactggaaaa tcccccttac 2760

gtgcccggcg tcgtgggcgg aacactgcag gccgctacaa tctgtgccag cagccaccag 2820

tttctgagca cccactacaa cctgcacaac ctgtacggcc tgaccgaggc cattgccagc 2880

caccgcgctc tcgtgaaagc cagaggcaca cggcccttcg tgatcagcag aagcaccttt 2940

gccggccacg gcagatacgc cggacattgg actggcgacg tgtggtcctc ttggggagcag 3000

ctggcctcta gcgtgcccga gatcctgcag ttcaatctgc tgggcgtgcc actcgtgggc 3060

gccgatgtgt gtggcttcct gggcaacacc tccgaggaac tgtgtgtgcg gtggacacag 3120

ctgggcgcct tctacccttt catgagaaac cacaacagcc tgctgagcct gccccaggaa 3180

ccctacagct ttagcgagcc tgcacagcag gccatgcgga aggccctgac actgagatac 3240

gctctgctgc cccacctgta caccctgttt caccaggccc atgtggccgg cgagacagtg 3300

gccagacctc tgtttctgga attccccaag gacagcagca cctggaccgt ggaccatcag 3360

ctgctgtggg gagaggctct gctgattacc ccagtgctgc aggcaggcaa ggccgaagtg 3420

accggctact ttcccctggg cacttggtac gacctgcaga ccgtgcctgt ggaagccctg 3480

ggatctctgc ctccacctcc tgccgctcct agagagcctg ccattcactc tgagggccag 3540

tgggtcacac tgcctgcccc cctggatacc atcaacgtgc acctgagggc cggctacatc 3600

ataccactgc agggacctgg cctgaccacc accgagtcta gacagcagcc aatggccctg 3660

gccgtggccc tgaccaaagg cggagaagct aggggcgagc tgttctggga cgatggcgag 3720

agcctggaag tgctggaaag aggcgcctat acccaagtga tcttcctggc ccggaacaac 3780

accatcgtga acgagctggt gcgcgtgacc tctgaaggcg ctggactgca gctgcagaaa 3840

gtgaccgtgc tgggagtggc cacagcccct cagcaggtgc tgtctaatgg cgtgcccgtg 3900

tccaacttca cctacagccc cgacaccaag gtgctggaca tctgcgtgtc actgctgatg 3960

ggagagcagt ttctggtgtc ctggtgctga ctcgagagat ctaccggtga attcaccgcg 4020

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4080

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4140

gcattgtctg agtaggtgtc attctattct gggggtggg gtgggggcta gctctaga 4198

<210> 27

<211> 917

<212> PRT

<213> Artificial sequence

<220>

<223> hGAA-Δ-8

<400> 27

Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val Leu Glu Glu

1 5 10 15

Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp

20 25 30

Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp

35 40 45

Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr

50 55 60

Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln

65 70 75 80

Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro

85 90 95

Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly

100 105 110

Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp

115 120 125

Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu

130 135 140

His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu

145 150 155 160

Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val

165 170 175

Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp

180 185 190

Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp

195 200 205

Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly

210 215 220

Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg

225 230 235 240

Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu

245 250 255

Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala

260 265 270

His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln

275 280 285

Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val

290 295 300

Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu

305 310 315 320

Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe

325 330 335

His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val

340 345 350

Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn

355 360 365

Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp

370 375 380

Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly

385 390 395 400

Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro

405 410 415

Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe

420 425 430

Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly

435 440 445

Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp

450 455 460

Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met

465 470 475 480

Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp

485 490 495

Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val

500 505 510

Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln

515 520 525

Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu

530 535 540

Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro

545 550 555 560

Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly

565 570 575

His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser

580 585 590

Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly

595 600 605

Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val

610 615 620

Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn

625 630 635 640

Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala

645 650 655

Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro

660 665 670

His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val

675 680 685

Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr

690 695 700

Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val

705 710 715 720

Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr

725 730 735

Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro

740 745 750

Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln

755 760 765

Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg

770 775 780

Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu

785 790 795 800

Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly

805 810 815

Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val

820 825 830

Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn

835 840 845

Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu

850 855 860

Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln

865 870 875 880

Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp

885 890 895

Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe

900 905 910

Leu Val Ser Trp Cys

915

<210> 28

<211> 883

<212> PRT

<213> Artificial sequence

<220>

<223> hGAA-Δ-42

<400> 28

Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro

1 5 10 15

Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu

20 25 30

Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu

35 40 45

Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr

50 55 60

Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr

65 70 75 80

Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu

85 90 95

Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe

100 105 110

Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr

115 120 125

Pro His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe

130 135 140

Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg

145 150 155 160

Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe

165 170 175

Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala

180 185 190

Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr

195 200 205

Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly

210 215 220

Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly

225 230 235 240

Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser

245 250 255

Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile

260 265 270

Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val

275 280 285

Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu

290 295 300

Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu

305 310 315 320

Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu

325 330 335

Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe

340 345 350

Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg

355 360 365

Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly

370 375 380

Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr

385 390 395 400

Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr

405 410 415

Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp

420 425 430

Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile

435 440 445

Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys

450 455 460

Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly

465 470 475 480

Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu

485 490 495

Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile

500 505 510

Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val

515 520 525

Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp

530 535 540

Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro

545 550 555 560

Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp

565 570 575

Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp

580 585 590

Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu

595 600 605

Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln

610 615 620

Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu

625 630 635 640

Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg

645 650 655

Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp

660 665 670

His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln

675 680 685

Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr

690 695 700

Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro

705 710 715 720

Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val

725 730 735

Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly

740 745 750

Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg

755 760 765

Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala

770 775 780

Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu

785 790 795 800

Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile

805 810 815

Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu

820 825 830

Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu

835 840 845

Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys

850 855 860

Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val

865 870 875 880

Ser Trp Cys

<210> 29

<211> 952

<212> PRT

<213> Homo sapiens

<400> 29

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 30

<211> 952

<212> PRT

<213> Homo sapiens

<400> 30

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 31

<211> 957

<212> PRT

<213> Homo sapiens

<400> 31

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Ala Arg Gly Pro Arg Val Leu Asp Ile Cys Val

930 935 940

Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

945 950 955

<210> 32

<211> 952

<212> PRT

<213> Homo sapiens

<400> 32

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Leu Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 33

<211> 925

<212> PRT

<213> Artificial sequence

<220>

<223> variant hGAAwt w/o sp

<400> 33

Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser

1 5 10 15

Gly Ser Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln Gly

20 25 30

Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro

35 40 45

Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp

50 55 60

Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly

65 70 75 80

Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly

85 90 95

Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu

100 105 110

Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr

115 120 125

Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val

130 135 140

Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala

145 150 155 160

Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His Ser Arg

165 170 175

Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly

180 185 190

Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr

195 200 205

Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser

210 215 220

Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu

225 230 235 240

Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu

245 250 255

Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu

260 265 270

Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser

275 280 285

Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg

290 295 300

Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro

305 310 315 320

Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met

325 330 335

Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser

340 345 350

Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His

355 360 365

Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg

370 375 380

Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met

385 390 395 400

Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp

405 410 415

Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp

420 425 430

Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro

435 440 445

Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr

450 455 460

Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His

465 470 475 480

Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser

485 490 495

Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu

500 505 510

Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala

515 520 525

Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu

530 535 540

His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu

545 550 555 560

Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe

565 570 575

Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser

580 585 590

Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn

595 600 605

Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly

610 615 620

Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe

625 630 635 640

Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu

645 650 655

Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu

660 665 670

Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln

675 680 685

Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe

690 695 700

Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly

705 710 715 720

Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val

725 730 735

Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro

740 745 750

Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu

755 760 765

Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu

770 775 780

Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln

785 790 795 800

Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu

805 810 815

Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp

820 825 830

Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln

835 840 845

Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg

850 855 860

Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu

865 870 875 880

Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val

885 890 895

Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val

900 905 910

Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

915 920 925

<210> 34

<211> 896

<212> PRT

<213> Artificial sequence

<220>

<223> hGAA-Δ-29

<400> 34

Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro

1 5 10 15

Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser

20 25 30

Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu

35 40 45

Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala

50 55 60

Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr

65 70 75 80

Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu

85 90 95

Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg

100 105 110

Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys

115 120 125

Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val

130 135 140

His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu

145 150 155 160

Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu

165 170 175

Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu

180 185 190

Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu

195 200 205

Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn

210 215 220

Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro

225 230 235 240

Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu

245 250 255

Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu

260 265 270

Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly

275 280 285

Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr

290 295 300

Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp

305 310 315 320

Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr

325 330 335

Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met

340 345 350

Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe

355 360 365

Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met

370 375 380

Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg

385 390 395 400

Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr

405 410 415

Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro

420 425 430

Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala

435 440 445

Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn

450 455 460

Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn

465 470 475 480

Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu

485 490 495

Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His

500 505 510

Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His

515 520 525

Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg

530 535 540

Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp

545 550 555 560

Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu

565 570 575

Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly

580 585 590

Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu

595 600 605

Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu

610 615 620

Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg

625 630 635 640

Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu

645 650 655

Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe

660 665 670

Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu

675 680 685

Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys

690 695 700

Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln

705 710 715 720

Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala

725 730 735

Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro

740 745 750

Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile

755 760 765

Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro

770 775 780

Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu

785 790 795 800

Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala

805 810 815

Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu

820 825 830

Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val

835 840 845

Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly

850 855 860

Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp

865 870 875 880

Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

885 890 895

<210> 35

<211> 882

<212> PRT

<213> Artificial sequence

<220>

<223> hGAA-Δ-43

<400> 35

His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro

1 5 10 15

Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln

20 25 30

Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln

35 40 45

Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro

50 55 60

Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala

65 70 75 80

Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr

85 90 95

Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr

100 105 110

Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro

115 120 125

His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser

130 135 140

Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val

145 150 155 160

Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu

165 170 175

Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu

180 185 190

His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu

195 200 205

Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser

210 215 220

His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val

225 230 235 240

Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro

245 250 255

Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe

260 265 270

Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val

275 280 285

Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys

290 295 300

Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn

305 310 315 320

Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp

325 330 335

Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg

340 345 350

Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr

355 360 365

Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser

370 375 380

Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn

385 390 395 400

Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala

405 410 415

Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met

420 425 430

Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp

435 440 445

Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro

450 455 460

Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly

465 470 475 480

Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser

485 490 495

Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala

500 505 510

Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile

515 520 525

Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr

530 535 540

Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu

545 550 555 560

Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val

565 570 575

Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr

580 585 590

Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu

595 600 605

Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala

610 615 620

Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr

625 630 635 640

Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro

645 650 655

Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His

660 665 670

Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala

675 680 685

Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp

690 695 700

Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro

705 710 715 720

Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr

725 730 735

Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr

740 745 750

Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln

755 760 765

Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg

770 775 780

Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg

785 790 795 800

Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val

805 810 815

Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln

820 825 830

Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser

835 840 845

Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val

850 855 860

Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser

865 870 875 880

tc

<210> 36

<211> 878

<212> PRT

<213> Artificial sequence

<220>

<223> hGAA-Δ-47

<400> 36

Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe

1 5 10 15

Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg

20 25 30

Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met

35 40 45

Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu

50 55 60

Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg

65 70 75 80

Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp

85 90 95

Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro

100 105 110

Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val His Ser

115 120 125

Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe

130 135 140

Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr

145 150 155 160

Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr

165 170 175

Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro

180 185 190

Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp

195 200 205

Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr

210 215 220

Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn

225 230 235 240

Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp

245 250 255

Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu

260 265 270

Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe

275 280 285

Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr

290 295 300

Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala

305 310 315 320

His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser

325 330 335

Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala

340 345 350

Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val

355 360 365

Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr

370 375 380

Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln

385 390 395 400

Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe

405 410 415

Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe

420 425 430

His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro

435 440 445

Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu

450 455 460

Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala

465 470 475 480

Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn

485 490 495

Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala

500 505 510

Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr

515 520 525

Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp

530 535 540

Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe

545 550 555 560

Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu

565 570 575

Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala

580 585 590

Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln

595 600 605

Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala

610 615 620

Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His

625 630 635 640

Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu

645 650 655

Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp

660 665 670

Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu

675 680 685

Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val

690 695 700

Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg

705 710 715 720

Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro Ala Pro

725 730 735

Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu

740 745 750

Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro Met Ala

755 760 765

Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe

770 775 780

Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr

785 790 795 800

Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val

805 810 815

Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val Thr Val

820 825 830

Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly Val Pro

835 840 845

Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp Ile Cys

850 855 860

Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

865 870 875

<210> 37

<211> 2745

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAwt-Δ-29

<400> 37

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccagcag 60

ggagccagca gaccagggcc ccgggatgcc caggcacacc ccgggcggcc gcgagcagtg 120

cccacacagt gcgacgtccc ccccaacagc cgcttcgatt gcgcccctga caaggccatc 180

acccaggaac agtgcgaggc ccgcggctgt tgctacatcc ctgcaaagca ggggctgcag 240

ggagcccaga tggggcagcc ctggtgcttc ttcccaccca gctaccccag ctacaagctg 300

gagaacctga gctcctctga aatgggctac acggccaccc tgacccgtac cacccccacc 360

ttcttcccca aggacatcct gaccctgcgg ctggacgtga tgatggagac tgagaaccgc 420

ctccacttca cgatcaaaga tccagctaac aggcgctacg aggtgccctt ggagaccccg 480

catgtccaca gccgggcacc gtccccactc tacagcgtgg agttctccga ggagcccttc 540

ggggtgatcg tgcgccggca gctggacggc cgcgtgctgc tgaacacgac ggtggcgccc 600

ctgttctttg cggaccagtt ccttcagctg tccacctcgc tgccctcgca gtatatcaca 660

ggcctcgccg agcacctcag tcccctgatg ctcagcacca gctggaccag gatcaccctg 720

tggaaccggg accttgcgcc cacgcccggt gcgaacctct acgggtctca ccctttctac 780

ctggcgctgg aggacggcgg gtcggcacac ggggtgttcc tgctaaacag caatgccatg 840

gatgtggtcc tgcagccgag ccctgccctt agctggaggt cgacaggtgg gatcctggat 900

gtctacatct tcctgggccc agagcccaag agcgtggtgc agcagtacct ggacgttgtg 960

ggatacccgt tcatgccgcc atactggggc ctgggcttcc acctgtgccg ctggggctac 1020

tcctccaccg ctatcacccg ccaggtggtg gagaacatga ccagggccca cttccccctg 1080

gacgtccagt ggaacgacct ggactacatg gactccccgga gggacttcac gttcaacaag 1140

gatggcttcc gggacttccc ggccatggtg caggagctgc accagggcgg ccggcgctac 1200

atgatgatcg tggatcctgc catcagcagc tcgggccctg ccggggagcta caggccctac 1260

gacgagggtc tgcggagggg ggttttcatc accaacgaga ccggccagcc gctgattggg 1320

aaggtatggc ccgggtccac tgccttcccc gacttcacca accccacagc cctggcctgg 1380

tgggaggaca tggtggctga gttccatgac caggtgccct tcgacggcat gtggattgac 1440

atgaacgagc cttccaactt catcaggggc tctgaggacg gctgccccaa caatgagctg 1500

gagaacccac cctacgtgcc tggggtggtt ggggggaccc tccaggcggc caccatctgt 1560

gcctccagcc accagtttct ctccacacac tacaacctgc acaacctcta cggcctgacc 1620

gaagccatcg cctccccacag ggcgctggtg aaggctcggg ggacacgccc atttgtgatc 1680

tcccgctcga cctttgctgg ccacggccga tacgccggcc actggacggg ggacgtgtgg 1740

agctcctggg agcagctcgc ctcctccgtg ccagaaatcc tgcagtttaa cctgctgggg 1800

gtgcctctgg tcggggccga cgtctgcggc ttcctgggca acacctcaga ggagctgtgt 1860

gtgcgctgga cccagctggg ggccttctac cccttcatgc ggaaccacaa cagcctgctc 1920

agtctgcccc aggagccgta cagcttcagc gagccggccc agcaggccat gaggaaggcc 1980

ctcaccctgc gctacgcact cctcccccac ctctacacac tgttccacca ggcccacgtc 2040

gcgggggaga ccgtggcccg gcccctcttc ctggagttcc ccaaggactc tagcacctgg 2100

actgtggacc accagctcct gtggggggag gccctgctca tcaccccagt gctccaggcc 2160

gggaaggccg aagtgactgg ctacttcccc ttgggcacat ggtacgacct gcagacggtg 2220

ccagtagagg cccttggcag cctcccaccc ccacctgcag ctccccgtga gccagccatc 2280

cacagcgagg ggcagtgggt gacgctgccg gcccccctgg acaccatcaa cgtccacctc 2340

cgggctgggt acatcatccc cctgcagggc cctggcctca caaccacaga gtcccgccag 2400

cagcccatgg ccctggctgt ggccctgacc aagggtgggg aggcccgagg ggagctgttc 2460

tgggacgatg gagagagcct ggaagtgctg gagcgagggg cctacacaca ggtcatcttc 2520

ctggccagga ataacacgat cgtgaatgag ctggtacgtg tgaccagtga gggagctggc 2580

ctgcagctgc agaaggtgac tgtcctgggc gtggccacgg cgccccagca ggtcctctcc 2640

aacggtgtcc ctgtctccaa cttcacctac agccccgaca ccaaggtcct ggacatctgt 2700

gtctcgctgt tgatgggaga gcagtttctc gtcagctggt gttag 2745

<210> 38

<211> 2745

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco1-Δ-29

<400> 38

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccagcag 60

ggcgcctcta gacctggacc tagagatgcc caggcccacc ccggcagacc tagagctgtg 120

cctacccagt gtgacgtgcc ccccaacagc agattcgact gcgcccctga caaggccatc 180

acccaggaac agtgcgaggc cagaggctgc tgctacatcc ctgccaagca gggactgcag 240

ggcgctcaga tgggacagcc ctggtgcttc ttcccaccct cctaccccag ctacaagctg 300

gaaaacctga gcagcagcga gatgggctac accgccaccc tgaccagaac cacccccaca 360

ttcttcccaa aggacatcct gaccctgcgg ctggacgtga tgatggaaac cgagaaccgg 420

ctgcacttca ccatcaagga ccccgccaat cggagatacg aggtgcccct ggaaaccccc 480

cacgtgcact ctagagcccc cagccctctg tacagcgtgg aattcagcga ggaacccttc 540

ggcgtgatcg tgcggagaca gctggatggc agagtgctgc tgaacacccac cgtggcccct 600

ctgttcttcg ccgaccagtt cctgcagctg agcaccagcc tgcccagcca gtacatcaca 660

ggactggccg agcacctgag ccccctgatg ctgagcacat cctggacccg gatcaccctg 720

tggaacaggg atctggcccc tacccctggc gccaatctgt acggcagcca ccctttctac 780

ctggccctgg aagatggcgg atctgcccac ggagtgtttc tgctgaactc caacgccatg 840

gacgtggtgc tgcagcctag ccctgccctg tcttggagaa gcacaggcgg catcctggat 900

gtgtacatct ttctgggccc cgagcccaag agcgtggtgc agcagtatct ggatgtcgtg 960

ggctacccct tcatgccccc ttactggggc ctgggattcc acctgtgcag atggggctac 1020

tccagcaccg ccatcaccag acaggtggtg gaaaacatga ccagagccca cttcccactg 1080

gatgtgcagt ggaacgacct ggactacatg gacagcagac gggacttcac cttcaacaag 1140

gacggcttcc gggacttccc cgccatggtg caggaactgc atcagggcgg cagacggtac 1200

atgatgatcg tggatcccgc catcagctcc tctggccctg ccggctctta cagaccctac 1260

gacgagggcc tgcggagagg cgtgttcatc accaacgaga caggccagcc cctgatcggc 1320

aaagtgtggc ctggcagcac agccttcccc gacttcacca atcctaccgc cctggcttgg 1380

tgggaggaca tggtggccga gttccacgac caggtgccct tcgacggcat gtggatcgac 1440

atgaacgagc ccagcaactt catccggggc agcgaggatg gctgccccaa caacgaactg 1500

gaaaatcccc cttacgtgcc cggcgtcgtg ggcggaacac tgcaggccgc tacaatctgt 1560

gccagcagcc accagtttct gagcacccac tacaacctgc acaacctgta cggcctgacc 1620

gaggccattg ccagccaccg cgctctcgtg aaagccagag gcacacggcc cttcgtgatc 1680

agcagaagca cctttgccgg ccacggcaga tacgccggac attggactgg cgacgtgtgg 1740

tcctcttggg agcagctggc ctctagcgtg cccgagatcc tgcagttcaa tctgctgggc 1800

gtgccactcg tgggcgccga tgtgtgtggc ttcctgggca acacctccga ggaactgtgt 1860

gtgcggtgga cacagctggg cgccttctac cctttcatga gaaaccacaa cagcctgctg 1920

agcctgcccc aggaacccta cagctttagc gagcctgcac agcaggccat gcggaaggcc 1980

ctgacactga gatacgctct gctgccccac ctgtacaccc tgtttcacca ggcccatgtg 2040

gccggcgaga cagtggccag acctctgttt ctggaattcc ccaaggacag cagcacctgg 2100

accgtggacc atcagctgct gtggggagag gctctgctga ttaccccagt gctgcaggca 2160

ggcaaggccg aagtgaccgg ctactttccc ctgggcactt ggtacgacct gcagaccgtg 2220

cctgtggaag ccctgggatc tctgcctcca cctcctgccg ctcctagaga gcctgccatt 2280

cactctgagg gccagtgggt cacactgcct gcccccctgg ataccatcaa cgtgcacctg 2340

agggccggct acatcatacc actgcaggga cctggcctga ccaccaccga gtctagacag 2400

cagccaatgg ccctggccgt ggccctgacc aaaggcggag aagctagggg cgagctgttc 2460

tgggacgatg gcgagagcct ggaagtgctg gaaagaggcg cctataccca agtgatcttc 2520

ctggccccgga acaacaccat cgtgaacgag ctggtgcgcg tgacctctga aggcgctgga 2580

ctgcagctgc agaaagtgac cgtgctggga gtggccacag cccctcagca ggtgctgtct 2640

aatggcgtgc ccgtgtccaa cttcacctac agccccgaca ccaaggtgct ggacatctgc 2700

gtgtcactgc tgatgggaga gcagtttctg gtgtcctggt gctga 2745

<210> 39

<211> 2745

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco2-Δ-29

<400> 39

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccaacag 60

ggagcttcca gaccaggacc gagagacgcc caagcccatc ctggtagacc aagagctgtg 120

cctacccaat gcgacgtgcc acccaactcc cgattcgact gcgcgccaga taaggctatt 180

acccaagagc agtgtgaagc cagaggttgc tgctacatcc cagcgaagca aggattgcaa 240

ggcgcccaaa tgggacaacc ttggtgtttc ttcccccctt cgtacccatc atataaactc 300

gaaaacctgt cctcttcgga aatgggttat actgccaccc tcaccagaac tactcctact 360

ttcttcccga aagacatctt gaccttgagg ctggacgtga tgatggagac tgaaaaccgg 420

ctgcatttca ctatcaaaga tcctgccaat cggcgatacg aggtccctct ggaaacccct 480

cacgtgcact cacgggctcc ttctccgctt tactccgtcg aattctctga ggaacccttc 540

ggagtgatcg ttagacgcca gctggatggt agagtgctgt tgaacactac tgtggcccca 600

cttttcttcg ctgaccagtt tctgcaactg tccacttccc tgccatccca gtacattact 660

ggactcgccg aacacctgtc gccactgatg ctctcgacct cttggactag aatcactttg 720

tggaacagag acttggcccc tactccggga gcaaatctgt acggaagcca ccctttttac 780

ctggcgctcg aagatggcgg atccgctcac ggagtgttcc tgctgaatag caacgcaatg 840

gacgtggtgc tgcaaccttc ccctgcactc agttggagaa gtaccggggg tattctggac 900

gtgtacatct tcctcggacc agaacccaag agcgtggtgc agcaatatct ggacgtggtc 960

ggataccctt ttatgcctcc ttactgggga ctgggattcc acctttgccg ttggggctac 1020

tcatccaccg ccattaccag acaggtggtg gagaatatga ccagagccca cttccctctc 1080

gacgtgcagt ggaacgatct ggactatatg gactccccgga gagatttcac cttcaacaag 1140

gacgggttcc gcgattttcc cgcgatggtt caagagctcc accagggtgg tcgaagatat 1200

atgatgatcg tcgacccagc catttcgagc agcggacccg ctggatctta tagaccttac 1260

gacgaaggcc ttaggagagg agtgttcatc acaaacgaga ctggacagcc tttgatcggt 1320

aaagtgtggc ctggatcaac cgcctttcct gactttacca atcccactgc cttggcttgg 1380

tgggaggaca tggtggccga attccacgac caagtcccctttgatggaat gtggatcgat 1440

atgaacgaac caagcaattt tatcagaggt tccgaagacg gttgccccaa caacgaactg 1500

gaaaaccctc cttatgtgcc cggagtcgtg ggcggaacat tacaggccgc gactatttgc 1560

gccagcagcc accaattcct gtccactcac tacaacctcc acaaccttta tggattaacc 1620

gaagctattg caagtcacag ggctctggtg aaggctagag ggactaggcc ctttgtgatc 1680

tcccgatcca cctttgccgg acacgggaga tacgccggtc actggactgg tgacgtgtgg 1740

agctcatggg aacaactggc ctcctccgtg ccggaaatct tacagttcaa ccttctgggt 1800

gtccctcttg tcggagcaga cgtgtgtggg tttcttggta acacctccga ggaactgtgt 1860

gtgcgctgga ctcaactggg tgcattctac ccattcatga gaaaccacaa ctccttgctg 1920

tccctgccac aagagcccta ctcgttcagc gagcctgcac aacaggctat gcggaaggca 1980

ctgaccctga gatacgccct gcttccacac ttatacactc tcttccatca agcgcatgtg 2040

gcaggagaaa ccgttgcaag gcctcttttc cttgaattcc ccaaggattc ctcgacttgg 2100

acggtggatc atcagctgct gtggggagaa gctctgctga ttactccagt gttgcaagcc 2160

ggaaaagctg aggtgaccgg atactttccg ctgggaacct ggtacgacct ccagactgtc 2220

cctgttgaag cccttggatc actgcctccg cctccggcag ctccacgcga accagctata 2280

cattccgagg gacagtgggt tacattacca gctcctctgg acacaatcaa cgtccactta 2340

agagctggct acattatccc tctgcaagga ccaggactga ctacgaccga gagcagacag 2400

cagccaatgg cactggctgt ggctctgacc aagggagggg aagctagagg agaactcttc 2460

tgggatgatg gggagtccct tgaagtgctg gaaagaggcg cttacactca agtcattttc 2520

cttgcacgga acaacaccat tgtgaacgaa ttggtgcgag tgaccagcga aggagctgga 2580

cttcaactgc agaaggtcac tgtgctcgga gtggctaccg ctcctcagca agtgctgtcg 2640

aatggagtcc ccgtgtcaaa ctttacctac tcccctgaca ctaaggtgct cgacatttgc 2700

gtgtccctcc tgatgggaga gcagttcctt gtgtcctggt gttga 2745

<210> 40

<211> 2706

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAwt-Δ-42

<400> 40

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcgcacac 60

cccgggcggc cgcgagcagt gcccacacag tgcgacgtcc cccccaacag ccgcttcgat 120

tgcgcccctg acaaggccat cacccaggaa cagtgcgagg cccgcggctg ttgctacatc 180

cctgcaaagc aggggctgca gggagcccag atggggcagc cctggtgctt cttcccaccc 240

agctacccca gctacaagct ggagaacctg agctcctctg aaatgggcta cacggccacc 300

ctgacccgta ccacccccac cttcttcccc aaggacatcc tgaccctgcg gctggacgtg 360

atgatggaga ctgagaaccg cctccacttc acgatcaaag atccagctaa caggcgctac 420

gaggtgccct tggagacccc gcatgtccac agccgggcac cgtccccact ctacagcgtg 480

gagttctccg aggagccctt cggggtgatc gtgcgccggc agctggacgg ccgcgtgctg 540

ctgaacacga cggtggcgcc cctgttcttt gcggaccagt tccttcagct gtccacctcg 600

ctgccctcgc agtatatcac aggcctcgcc gagcacctca gtcccctgat gctcagcacc 660

agctggacca ggatcaccct gtggaaccgg gaccttgcgc ccacgcccgg tgcgaacctc 720

tacgggtctc accctttcta cctggcgctg gaggacggcg ggtcggcaca cggggtgttc 780

ctgctaaaca gcaatgccat ggatgtggtc ctgcagccga gccctgccct tagctggagg 840

tcgacaggtg ggatcctgga tgtctacatc ttcctgggcc cagagcccaa gagcgtggtg 900

cagcagtacc tggacgttgt gggatacccg ttcatgccgc catactgggg cctgggcttc 960

cacctgtgcc gctggggcta ctcctccacc gctatcaccc gccaggtggt ggagaacatg 1020

accagggccc acttccccct ggacgtccag tggaacgacc tggactacat ggactcccgg 1080

agggacttca cgttcaacaa ggatggcttc cgggacttcc cggccatggt gcaggagctg 1140

caccagggcg gccggcgcta catgatgatc gtggatcctg ccatcagcag ctcgggccct 1200

gccgggagct acaggcccta cgacgagggt ctgcggaggg gggttttcat caccaacgag 1260

accggccagc cgctgattgg gaaggtatgg cccgggtcca ctgccttccc cgacttcacc 1320

aaccccacag ccctggcctg gtgggaggac atggtggctg agttccatga ccaggtgccc 1380

ttcgacggca tgtggattga catgaacgag ccttccaact tcatcagggg ctctgaggac 1440

ggctgcccca acaatgagct ggagaaccca ccctacgtgc ctggggtggt tggggggacc 1500

ctccaggcgg ccaccatctg tgcctccagc caccagtttc tctccacaca ctacaacctg 1560

cacaacctct acggcctgac cgaagccatc gcctcccaca gggcgctggt gaaggctcgg 1620

gggacacgcc catttgtgat ctcccgctcg acctttgctg gccacggccg atacgccggc 1680

cactggacgg gggacgtgtg gagctcctgg gagcagctcg cctcctccgt gccagaaatc 1740

ctgcagttta acctgctggg ggtgcctctg gtcggggccg acgtctgcgg cttcctgggc 1800

aacacctcag aggagctgtg tgtgcgctgg acccagctgg gggccttcta ccccttcatg 1860

cggaaccaca acagcctgct cagtctgccc caggagccgt acagcttcag cgagccggcc 1920

cagcaggcca tgaggaaggc cctcaccctg cgctacgcac tcctccccca cctctacaca 1980

ctgttccacc aggcccacgt cgcgggggag accgtggccc ggcccctctt cctggagttc 2040

cccaaggact ctagcacctg gactgtggac caccagctcc tgtgggggga ggccctgctc 2100

atcaccccag tgctccaggc cgggaaggcc gaagtgactg gctacttccc cttgggcaca 2160

tggtacgacc tgcagacggt gccagtagag gcccttggca gcctcccacc cccacctgca 2220

gctccccgtg agccagccat ccacagcgag gggcagtggg tgacgctgcc ggcccccctg 2280

gacaccatca acgtccacct ccgggctggg tacatcatcc ccctgcaggg ccctggcctc 2340

acaaccacag agtcccgcca gcagcccatg gccctggctg tggccctgac caagggtggg 2400

gaggcccgag gggagctgtt ctgggacgat ggagagagcc tggaagtgct ggagcgaggg 2460

gcctacacac aggtcatctt cctggccagg aataacacga tcgtgaatga gctggtacgt 2520

gtgaccagtg agggagctgg cctgcagctg cagaaggtga ctgtcctggg cgtggccacg 2580

gcgccccagc aggtcctctc caacggtgtc cctgtctcca acttcaccta cagccccgac 2640

accaaggtcc tggacatctg tgtctcgctg ttgatggggag agcagtttct cgtcagctgg 2700

tgttag 2706

<210> 41

<211> 2706

<212> DNA

<213> Artificial sequence

<220>

<223> sp7-hGAAco2-Δ-42

<400> 41

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcgcccat 60

cctggtagac caagagctgt gcctacccaa tgcgacgtgc cacccaactc ccgattcgac 120

tgcgcgccag ataaggctat tacccaagag cagtgtgaag ccagaggttg ctgctacatc 180

ccagcgaagc aaggattgca aggcgcccaa atgggacaac cttggtgttt cttcccccct 240

tcgtacccat catataaact cgaaaacctg tcctcttcgg aaatgggtta tactgccacc 300

ctcaccagaa ctactcctac tttcttcccg aaagacatct tgaccttgag gctggacgtg 360

atgatggaga ctgaaaaccg gctgcatttc actatcaaag atcctgccaa tcggcgatac 420

gaggtccctc tggaaacccc tcacgtgcac tcacgggctc cttctccgct ttactccgtc 480

gaattctctg aggaaccctt cggagtgatc gttagacgcc agctggatgg tagagtgctg 540

ttgaacacta ctgtggcccc acttttcttc gctgaccagt ttctgcaact gtccacttcc 600

ctgccatccc agtacattac tggactcgcc gaacacctgt cgccactgat gctctcgacc 660

tcttggacta gaatcacttt gtggaacaga gacttggccc ctactccggg agcaaatctg 720

tacggaagcc acccttttta cctggcgctc gaagatggcg gatccgctca cggagtgttc 780

ctgctgaata gcaacgcaat ggacgtggtg ctgcaacctt cccctgcact cagttggaga 840

agtaccgggg gtattctgga cgtgtacatc ttcctcggac cagaacccaa gagcgtggtg 900

cagcaatatc tggacgtggt cggataccct tttatgcctc cttactgggg actgggattc 960

cacctttgcc gttggggcta ctcatccacc gccattacca gacaggtggt ggagaatatg 1020

accagagccc acttccctct cgacgtgcag tggaacgatc tggactatat ggactcccgg 1080

agagatttca ccttcaacaa ggacgggttc cgcgattttc ccgcgatggt tcaagagctc 1140

caccagggtg gtcgaagata tatgatgatc gtcgacccag ccatttcgag cagcggaccc 1200

gctggatctt atagacctta cgacgaaggc cttaggagag gagtgttcat cacaaacgag 1260

actggacagc ctttgatcgg taaagtgtgg cctggatcaa ccgcctttcc tgactttacc 1320

aatcccactg ccttggcttg gtgggaggac atggtggccg aattccacga ccaagtcccc 1380

tttgatggaa tgtggatcga tatgaacgaa ccaagcaatt ttatcagagg ttccgaagac 1440

ggttgcccca acaacgaact ggaaaaccct ccttatgtgc ccggagtcgt gggcggaaca 1500

ttacaggccg cgactatttg cgccagcagc caccaattcc tgtccactca ctacaacctc 1560

cacaaccttt atggattaac cgaagctatt gcaagtcaca gggctctggt gaaggctaga 1620

gggactaggc cctttgtgat ctcccgatcc acctttgccg gacacgggag atacgccggt 1680

cactggactg gtgacgtgtg gagctcatgg gaacaactgg cctcctccgt gccggaaatc 1740

ttacagttca accttctggg tgtccctctt gtcggagcag acgtgtgtgg gtttcttggt 1800

aacacctccg aggaactgtg tgtgcgctgg actcaactgg gtgcattcta cccattcatg 1860

agaaaccaca actccttgct gtccctgcca caagagccct actcgttcag cgagcctgca 1920

caacaggcta tgcggaaggc actgaccctg agatacgccc tgcttccaca cttatacact 1980

ctcttccatc aagcgcatgt ggcaggagaa accgttgcaa ggcctctttt ccttgaattc 2040

cccaaggatt cctcgacttg gacggtggat catcagctgc tgtggggaga agctctgctg 2100

attactccag tgttgcaagc cggaaaagct gaggtgaccg gatactttcc gctgggaacc 2160

tggtacgacc tccagactgt ccctgttgaa gcccttggat cactgcctcc gcctccggca 2220

gctccacgcg aaccagctat acattccgag ggacagtggg ttacattacc agctcctctg 2280

gacacaatca acgtccactt aagagctggc tacattatcc ctctgcaagg accaggactg 2340

actacgaccg agagcagaca gcagccaatg gcactggctg tggctctgac caagggaggg 2400

gaagctagag gagaactctt ctgggatgat ggggagtccc ttgaagtgct ggaaagaggc 2460

gcttacactc aagtcatttt ccttgcacgg aacaacacca ttgtgaacga attggtgcga 2520

gtgaccagcg aaggagctgg acttcaactg cagaaggtca ctgtgctcgg agtggctacc 2580

gctcctcagc aagtgctgtc gaatggagtc cccgtgtcaa actttaccta ctcccctgac 2640

actaaggtgc tcgacatttg cgtgtccctc ctgatgggag agcagttcct tgtgtcctgg 2700

tgttga 2706

<210> 42

<211> 2703

<212> DNA

<213> Artificial sequence

<220>

<223> sp7-hGAAwt-Δ-43

<400> 42

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccaccc 60

gggcggccgc gagcagtgcc cacacagtgc gacgtccccc ccaacagccg cttcgattgc 120

gcccctgaca aggccatcac ccaggaacag tgcgaggccc gcggctgttg ctacatccct 180

gcaaagcagg ggctgcaggg agcccagatg gggcagccct ggtgcttctt cccacccagc 240

taccccagct acaagctgga gaacctgagc tcctctgaaa tgggctacac ggccaccctg 300

acccgtacca cccccacctt cttccccaag gacatcctga ccctgcggct ggacgtgatg 360

atggagactg agaaccgcct ccacttcacg atcaaagatc cagctaacag gcgctacgag 420

gtgcccttgg agaccccgca tgtccacagc cgggcaccgt ccccactcta cagcgtggag 480

ttctccgagg agcccttcgg ggtgatcgtg cgccggcagc tggacggccg cgtgctgctg 540

aacacgacgg tggcgcccct gttctttgcg gaccagttcc ttcagctgtc cacctcgctg 600

ccctcgcagt atatcacagg cctcgccgag cacctcagtc ccctgatgct cagcaccagc 660

tggaccagga tcaccctgtg gaaccgggac cttgcgccca cgcccggtgc gaacctctac 720

gggtctcacc ctttctacct ggcgctggag gacggcgggt cggcacacgg ggtgttcctg 780

ctaaacagca atgccatgga tgtggtcctg cagccgagcc ctgcccttag ctggaggtcg 840

acaggtggga tcctggatgt ctacatcttc ctgggcccag agcccaagag cgtggtgcag 900

cagtacctgg acgttgtggg atacccgttc atgccgccat actggggcct gggcttccac 960

ctgtgccgct ggggctactc ctccaccgct atcacccgcc aggtggtgga gaacatgacc 1020

agggcccact tccccctgga cgtccagtgg aacgacctgg actacatgga ctcccggagg 1080

gacttcacgt tcaacaagga tggcttccgg gacttcccgg ccatggtgca ggagctgcac 1140

cagggcggcc ggcgctacat gatgatcgtg gatcctgcca tcagcagctc gggccctgcc 1200

gggagctaca ggccctacga cgagggtctg cggagggggg ttttcatcac caacgagacc 1260

ggccagccgc tgattgggaa ggtatggccc gggtccactg ccttccccga cttcaccaac 1320

cccacagccc tggcctggtg ggaggacatg gtggctgagt tccatgacca ggtgcccttc 1380

gacggcatgt ggattgacat gaacgagcct tccaacttca tcaggggctc tgaggacggc 1440

tgccccaaca atgagctgga gaacccaccc tacgtgcctg gggtggttgg ggggaccctc 1500

caggcggcca ccatctgtgc ctccagccac cagtttctct ccacacacta caacctgcac 1560

aacctctacg gcctgaccga agccatcgcc tcccacaggg cgctggtgaa ggctcggggg 1620

acacgcccat ttgtgatctc ccgctcgacc tttgctggcc acggccgata cgccggccac 1680

tggacggggg acgtgtggag ctcctggggag cagctcgcct cctccgtgcc agaaatcctg 1740

cagtttaacc tgctgggggt gcctctggtc ggggccgacg tctgcggctt cctgggcaac 1800

acctcagagg agctgtgtgt gcgctggacc cagctggggg ccttctaccc cttcatgcgg 1860

aaccacaaca gcctgctcag tctgccccag gagccgtaca gcttcagcga gccggcccag 1920

caggccatga ggaaggcct caccctgcgc tacgcactcc tcccccacct ctacacactg 1980

ttccaccagg cccacgtcgc gggggagacc gtggcccggc ccctcttcct ggagttcccc 2040

aaggactcta gcacctggac tgtggaccac cagctcctgt ggggggaggc cctgctcatc 2100

accccagtgc tccaggccgg gaaggccgaa gtgactggct acttcccctt gggcacatgg 2160

tacgacctgc agacggtgcc agtagaggcc cttggcagcc tcccacccccc acctgcagct 2220

ccccgtgagc cagccatcca cagcgagggg cagtgggtga cgctgccggc ccccctggac 2280

accatcaacg tccacctccg ggctgggtac atcatccccc tgcagggccc tggcctcaca 2340

accacagagt cccgccagca gcccatggcc ctggctgtgg ccctgaccaa gggtggggag 2400

gcccgagggg agctgttctg ggacgatgga gagagcctgg aagtgctgga gcgaggggcc 2460

tacacacagg tcatcttcct ggccaggaat aacacgatcg tgaatgagct ggtacgtgtg 2520

accagtgagg gagctggcct gcagctgcag aaggtgactg tcctgggcgt ggccacggcg 2580

ccccagcagg tcctctccaa cggtgtccct gtctccaact tcacctacag ccccgacacc 2640

aaggtcctgg acatctgtgt ctcgctgttg atgggagagc agtttctcgt cagctggtgt 2700

tag 2703

<210> 43

<211> 2703

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco1-Δ-43

<400> 43

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccaccc 60

ggcagaccta gagctgtgcc tacccagtgt gacgtgcccc ccaacagcag attcgactgc 120

gcccctgaca aggccatcac ccaggaacag tgcgaggcca gaggctgctg ctacatccct 180

gccaagcagg gactgcaggg cgctcagatg ggacagccct ggtgcttctt cccaccctcc 240

taccccagct acaagctgga aaacctgagc agcagcgaga tgggctacac cgccaccctg 300

accagaacca cccccacatt cttcccaaag gacatcctga ccctgcggct ggacgtgatg 360

atggaaaccg agaaccggct gcacttcacc atcaaggacc ccgccaatcg gagatacgag 420

gtgcccctgg aaacccccca cgtgcactct agagccccca gccctctgta cagcgtggaa 480

ttcagcgagg aacccttcgg cgtgatcgtg cggagacagc tggatggcag agtgctgctg 540

aacaccaccg tggcccctct gttcttcgcc gaccagttcc tgcagctgag caccagcctg 600

cccagccagt acatcacagg actggccgag cacctgagcc ccctgatgct gagcacatcc 660

tggacccgga tcaccctgtg gaacagggat ctggccccta cccctggcgc caatctgtac 720

ggcagccacc ctttctacct ggccctggaa gatggcggat ctgcccacgg agtgtttctg 780

ctgaactcca acgccatgga cgtggtgctg cagcctagcc ctgccctgtc ttggagaagc 840

acaggcggca tcctggatgt gtacatcttt ctgggccccg agcccaagag cgtggtgcag 900

cagtatctgg atgtcgtggg ctaccccttc atgccccctt actggggcct gggattccac 960

ctgtgcagat ggggctactc cagcaccgcc atcaccagac aggtggtgga aaacatgacc 1020

agagcccact tcccactgga tgtgcagtgg aacgacctgg actacatgga cagcagacgg 1080

gacttcacct tcaacaagga cggcttccgg gacttccccg ccatggtgca ggaactgcat 1140

cagggcggca gacggtacat gatgatcgtg gatcccgcca tcagctcctc tggccctgcc 1200

ggctcttaca gaccctacga cgagggcctg cggagaggcg tgttcatcac caacgagaca 1260

ggccagcccc tgatcggcaa agtgtggcct ggcagcacag ccttccccga cttcaccaat 1320

cctaccgccc tggcttggtg ggaggacatg gtggccgagt tccacgacca ggtgcccttc 1380

gacggcatgt ggatcgacat gaacgagccc agcaacttca tccggggcag cgaggatggc 1440

tgccccaaca acgaactgga aaatccccct tacgtgcccg gcgtcgtggg cggaacactg 1500

caggccgcta caatctgtgc cagcagccac cagtttctga gcacccacta caacctgcac 1560

aacctgtacg gcctgaccga ggccattgcc agccaccgcg ctctcgtgaa agccagaggc 1620

acacggccct tcgtgatcag cagaagcacc tttgccggcc acggcagata cgccggacat 1680

tggactggcg acgtgtggtc ctcttggggag cagctggcct ctagcgtgcc cgagatcctg 1740

cagttcaatc tgctgggcgt gccactcgtg ggcgccgatg tgtgtggctt cctgggcaac 1800

acctccgagg aactgtgtgt gcggtggaca cagctgggcg ccttctaccc tttcatgaga 1860

aaccacaaca gcctgctgag cctgccccag gaaccctaca gctttagcga gcctgcacag 1920

caggccatgc ggaaggcct gacactgaga tacgctctgc tgccccacct gtacaccctg 1980

tttcaccagg cccatgtggc cggcgagaca gtggccagac ctctgtttct ggaattcccc 2040

aaggacagca gcacctggac cgtggaccat cagctgctgt ggggagaggc tctgctgatt 2100

accccagtgc tgcaggcagg caaggccgaa gtgaccggct actttcccct gggcacttgg 2160

tacgacctgc agaccgtgcc tgtggaagcc ctgggatctc tgcctccacc tcctgccgct 2220

cctagagagc ctgccattca ctctgagggc cagtgggtca cactgcctgc ccccctggat 2280

accatcaacg tgcacctgag ggccggctac atcataccac tgcagggacc tggcctgacc 2340

accaccgagt ctagacagca gccaatggcc ctggccgtgg ccctgaccaa aggcggagaa 2400

gctaggggcg agctgttctg ggacgatggc gagagcctgg aagtgctgga aagaggcgcc 2460

tatacccaag tgatcttcct ggcccggaac aacaccatcg tgaacgagct ggtgcgcgtg 2520

acctctgaag gcgctggact gcagctgcag aaagtgaccg tgctgggagt ggccacagcc 2580

cctcagcagg tgctgtctaa tggcgtgccc gtgtccaact tcacctacag ccccgacacc 2640

aaggtgctgg acatctgcgt gtcactgctg atgggagagc agtttctggt gtcctggtgc 2700

tga 2703

<210> 44

<211> 2703

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco2-Δ-43

<400> 44

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccatcct 60

ggtagaccaa gagctgtgcc tacccaatgc gacgtgccac ccaactcccg attcgactgc 120

gcgccagata aggctattac ccaagagcag tgtgaagcca gaggttgctg ctacatccca 180

gcgaagcaag gattgcaagg cgcccaaatg ggacaacctt ggtgtttctt ccccccttcg 240

tacccatcat ataaactcga aaacctgtcc tcttcggaaa tgggttatac tgccaccctc 300

accagaacta ctcctacttt cttcccgaaa gacatcttga ccttgaggct ggacgtgatg 360

atggagactg aaaaccggct gcatttcact atcaaagatc ctgccaatcg gcgatacgag 420

gtccctctgg aaacccctca cgtgcactca cgggctcctt ctccgcttta ctccgtcgaa 480

ttctctgagg aacccttcgg agtgatcgtt agacgccagc tggatggtag agtgctgttg 540

aacactactg tggccccact tttcttcgct gaccagtttc tgcaactgtc cacttccctg 600

ccatcccagt acattactgg actcgccgaa cacctgtcgc cactgatgct ctcgacctct 660

tggactagaa tcactttgtg gaacagagac ttggccccta ctccgggagc aaatctgtac 720

ggaagccacc ctttttacct ggcgctcgaa gatggcggat ccgctcacgg agtgttcctg 780

ctgaatagca acgcaatgga cgtggtgctg caaccttccc ctgcactcag ttggagaagt 840

accgggggta ttctggacgt gtacatcttc ctcggaccag aacccaagag cgtggtgcag 900

caatatctgg acgtggtcgg ataccctttt atgcctcctt actggggact gggattccac 960

ctttgccgtt ggggctactc atccaccgcc attaccagac aggtggtgga gaatatgacc 1020

agagcccact tccctctcga cgtgcagtgg aacgatctgg actatatgga ctcccggaga 1080

gatttcacct tcaacaagga cgggttccgc gattttcccg cgatggttca agagctccac 1140

cagggtggtc gaagatatat gatgatcgtc gacccagcca tttcgagcag cggacccgct 1200

ggatcttata gaccttacga cgaaggcctt aggagaggag tgttcatcac aaacgagact 1260

ggacagcctt tgatcggtaa agtgtggcct ggatcaaccg cctttcctga ctttaccaat 1320

cccactgcct tggcttggtg ggaggacatg gtggccgaat tccacgacca agtccccttt 1380

gatggaatgt ggatcgatat gaacgaacca agcaatttta tcagaggttc cgaagacggt 1440

tgccccaaca acgaactgga aaaccctcct tatgtgcccg gagtcgtggg cggaacatta 1500

caggccgcga ctatttgcgc cagcagccac caattcctgt ccactcacta caacctccac 1560

aacctttatg gattaaccga agctattgca agtcacaggg ctctggtgaa ggctagaggg 1620

actaggccct ttgtgatctc ccgatccacc tttgccggac acggggagata cgccggtcac 1680

tggactggtg acgtgtggag ctcatgggaa caactggcct cctccgtgcc ggaaatctta 1740

cagttcaacc ttctgggtgt ccctcttgtc ggagcagacg tgtgtgggtt tcttggtaac 1800

acctccgagg aactgtgtgt gcgctggact caactgggtg cattctaccc attcatgaga 1860

aaccacaact ccttgctgtc cctgccacaa gagccctact cgttcagcga gcctgcacaa 1920

caggctatgc ggaaggcact gaccctgaga tacgccctgc ttccacactt atacactctc 1980

ttccatcaag cgcatgtggc aggagaaacc gttgcaaggc ctcttttcct tgaattcccc 2040

aaggattcct cgacttggac ggtggatcat cagctgctgt ggggagaagc tctgctgatt 2100

actccagtgt tgcaagccgg aaaagctgag gtgaccggat actttccgct gggaacctgg 2160

tacgacctcc agactgtccc tgttgaagcc cttggatcac tgcctccgcc tccggcagct 2220

ccacgcgaac cagctataca ttccgaggga cagtgggtta cattaccagc tcctctggac 2280

acaatcaacg tccacttaag agctggctac attatccctc tgcaaggacc aggactgact 2340

acgaccgaga gcagacagca gccaatggca ctggctgtgg ctctgaccaa gggaggggaa 2400

gctagaggag aactcttctg ggatgatggg gagtcccttg aagtgctgga aagaggcgct 2460

tacactcaag tcattttcct tgcacggaac aacaccattg tgaacgaatt ggtgcgagtg 2520

accagcgaag gagctggact tcaactgcag aaggtcactg tgctcggagt ggctaccgct 2580

cctcagcaag tgctgtcgaa tggagtcccc gtgtcaaact ttacctactc ccctgacact 2640

aaggtgctcg acatttgcgt gtccctcctg atgggagagc agttccttgt gtcctggtgt 2700

tga 2703

<210> 45

<211> 2691

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAwt-Δ-47

<400> 45

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcccgcga 60

gcagtgccca cacagtgcga cgtccccccc aacagccgct tcgattgcgc ccctgacaag 120

gccatcaccc aggaacagtg cgaggcccgc ggctgttgct acatccctgc aaagcagggg 180

ctgcagggag cccagatggg gcagccctgg tgcttcttcc cacccagcta ccccagctac 240

aagctggaga acctgagctc ctctgaaatg ggctacacgg ccaccctgac ccgtaccacc 300

cccaccttct tccccaagga catcctgacc ctgcggctgg acgtgatgat ggagactgag 360

aaccgcctcc acttcacgat caaagatcca gctaacaggc gctacgaggt gcccttggag 420

accccgcatg tccacagccg ggcaccgtcc ccactctaca gcgtggagtt ctccgaggag 480

cccttcgggg tgatcgtgcg ccggcagctg gacggccgcg tgctgctgaa cacgacggtg 540

gcgcccctgt tctttgcgga ccagttcctt cagctgtcca cctcgctgcc ctcgcagtat 600

atcacaggcc tcgccgagca cctcagtccc ctgatgctca gcaccagctg gaccaggatc 660

accctgtgga accgggacct tgcgcccacg cccggtgcga acctctacgg gtctcaccct 720

ttctacctgg cgctggagga cggcgggtcg gcacacgggg tgttcctgct aaacagcaat 780

gccatggatg tggtcctgca gccgagccct gcccttagct ggaggtcgac aggtggggatc 840

ctggatgtct acatcttcct gggcccagag cccaagagcg tggtgcagca gtacctggac 900

gttgtggggat acccgttcat gccgccatac tggggcctgg gcttccacct gtgccgctgg 960

ggctactcct ccaccgctat cacccgccag gtggtggaga acatgaccag ggcccacttc 1020

cccctggacg tccagtggaa cgacctggac tacatggact cccggaggga cttcacgttc 1080

aacaaggatg gcttccggga cttcccggcc atggtgcagg agctgcacca gggcggccgg 1140

cgctacatga tgatcgtgga tcctgccatc agcagctcgg gccctgccgg gagctacagg 1200

ccctacgacg agggtctgcg gaggggggttttcatcacca acgagaccgg ccagccgctg 1260

attgggaagg tatggcccgg gtccactgcc ttccccgact tcaccaaccc cacagccctg 1320

gcctggtggg aggacatggt ggctgagttc catgaccagg tgcccttcga cggcatgtgg 1380

attgacatga acgagccttc caacttcatc aggggctctg aggacggctg ccccaacaat 1440

gagctggaga acccacccta cgtgcctggg gtggttgggg ggaccctcca ggcggccacc 1500

atctgtgcct ccagccacca gtttctctcc acacactaca acctgcacaa cctctacggc 1560

ctgaccgaag ccatcgcctc ccacagggcg ctggtgaagg ctcgggggac acgcccattt 1620

gtgatctccc gctcgacctt tgctggccac ggccgatacg ccggccactg gacgggggac 1680

gtgtggagct cctgggagca gctcgcctcc tccgtgccag aaatcctgca gtttaacctg 1740

ctgggggtgc ctctggtcgg ggccgacgtc tgcggcttcc tgggcaacac ctcagaggag 1800

ctgtgtgtgc gctggaccca gctgggggcc ttctacccct tcatgcggaa ccacaacagc 1860

ctgctcagtc tgccccagga gccgtacagc ttcagcgagc cggcccagca ggccatgagg 1920

aaggccctca ccctgcgcta cgcactcctc ccccacctct acacactgtt ccaccaggcc 1980

cacgtcgcgg gggagaccgt ggcccggccc ctcttcctgg agttccccaa ggactctagc 2040

acctggactg tggaccacca gctcctgtgg ggggaggccc tgctcatcac cccagtgctc 2100

caggccggga aggccgaagt gactggctac ttccccttgg gcacatggta cgacctgcag 2160

acggtgccag tagaggccct tggcagcctc ccacccccac ctgcagctcc ccgtgagcca 2220

gccatccaca gcgaggggca gtgggtgacg ctgccggccc ccctggacac catcaacgtc 2280

cacctccggg ctgggtacat catccccctg cagggccctg gcctcacaac cacagagtcc 2340

cgccagcagc ccatggccct ggctgtggcc ctgaccaagg gtggggaggc ccgaggggag 2400

ctgttctggg acgatggaga gagcctggaa gtgctggagc gaggggccta cacacaggtc 2460

atcttcctgg ccaggaataa cacgatcgtg aatgagctgg tacgtgtgac cagtgaggga 2520

gctggcctgc agctgcagaa ggtgactgtc ctgggcgtgg ccacggcgcc ccagcaggtc 2580

ctctccaacg gtgtccctgt ctccaacttc acctacagcc ccgacaccaa ggtcctggac 2640

atctgtgtct cgctgttgat gggagagcag tttctcgtca gctggtgtta g 2691

<210> 46

<211> 2691

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco1-Δ-47

<400> 46

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccctaga 60

gctgtgccta cccagtgtga cgtgcccccc aacagcagat tcgactgcgc ccctgacaag 120

gccatcaccc aggaacagtg cgaggccaga ggctgctgct acatccctgc caagcaggga 180

ctgcagggcg ctcagatggg acagccctgg tgcttcttcc caccctccta ccccagctac 240

aagctggaaa acctgagcag cagcgagatg ggctacaccg ccaccctgac cagaaccacc 300

cccacattct tcccaaagga catcctgacc ctgcggctgg acgtgatgat ggaaaccgag 360

aaccggctgc acttcaccat caaggacccc gccaatcgga gatacgaggt gcccctggaa 420

accccccacg tgcactctag agcccccagc cctctgtaca gcgtggaatt cagcgaggaa 480

cccttcggcg tgatcgtgcg gagacagctg gatggcagag tgctgctgaa caccaccgtg 540

gcccctctgt tcttcgccga ccagttcctg cagctgagca ccagcctgcc cagccagtac 600

atcacaggac tggccgagca cctgagcccc ctgatgctga gcacatcctg gacccggatc 660

accctgtgga acagggatct ggcccctacc cctggcgcca atctgtacgg cagccaccct 720

ttctacctgg ccctggaaga tggcggatct gcccacggag tgtttctgct gaactccaac 780

gccatggacg tggtgctgca gcctagccct gccctgtctt ggagaagcac aggcggcatc 840

ctggatgtgt acatctttct gggccccgag cccaagagcg tggtgcagca gtatctggat 900

gtcgtgggct accccttcat gcccccttac tggggcctgg gattccacct gtgcagatgg 960

ggctactcca gcaccgccat caccagacag gtggtggaaa acatgaccag agcccacttc 1020

ccactggatg tgcagtggaa cgacctggac tacatggaca gcagacggga cttcaccttc 1080

aacaaggacg gcttccggga cttccccgcc atggtgcagg aactgcatca gggcggcaga 1140

cggtacatga tgatcgtgga tcccgccatc agctcctctg gccctgccgg ctcttacaga 1200

ccctacgacg agggcctgcg gagaggcgtg ttcatcacca acgagacagg ccagcccctg 1260

atcggcaaag tgtggcctgg cagcacagcc ttccccgact tcaccaatcc taccgccctg 1320

gcttggtggg aggacatggt ggccgagttc cacgaccagg tgcccttcga cggcatgtgg 1380

atcgacatga acgagcccag caacttcatc cggggcagcg aggatggctg ccccaacaac 1440

gaactggaaa atccccctta cgtgcccggc gtcgtgggcg gaacactgca ggccgctaca 1500

atctgtgcca gcagccacca gtttctgagc accccactaca acctgcacaa cctgtacggc 1560

ctgaccgagg ccattgccag ccaccgcgct ctcgtgaaag ccagaggcac acggcccttc 1620

gtgatcagca gaagcacctt tgccggccac ggcagatacg ccggacattg gactggcgac 1680

gtgtggtcct cttgggagca gctggcctct agcgtgcccg agatcctgca gttcaatctg 1740

ctgggcgtgc cactcgtggg cgccgatgtg tgtggcttcc tgggcaacac ctccgaggaa 1800

ctgtgtgtgc ggtggacaca gctgggcgcc ttctaccctt tcatgagaaa ccacaacagc 1860

ctgctgagcc tgccccagga accctacagc tttagcgagc ctgcacagca ggccatgcgg 1920

aaggccctga cactgagata cgctctgctg ccccacctgt acaccctgtt tcaccaggcc 1980

catgtggccg gcgagacagt ggccagacct ctgtttctgg aattccccaa ggacagcagc 2040

acctggaccg tggaccatca gctgctgtgg ggagaggctc tgctgattac cccagtgctg 2100

caggcaggca aggccgaagt gaccggctac tttcccctgg gcacttggta cgacctgcag 2160

accgtgcctg tggaagccct gggatctctg cctccacctc ctgccgctcc tagagagcct 2220

gccattcact ctgagggcca gtgggtcaca ctgcctgccc ccctggatac catcaacgtg 2280

cacctgaggg ccggctacat cataccactg cagggacctg gcctgaccac caccgagtct 2340

agacagcagc caatggccct ggccgtggcc ctgaccaaag gcggagaagc taggggcgag 2400

ctgttctggg acgatggcga gagcctggaa gtgctggaaa gaggcgccta tacccaagtg 2460

atcttcctgg cccggaacaa caccatcgtg aacgagctgg tgcgcgtgac ctctgaaggc 2520

gctggactgc agctgcagaa agtgaccgtg ctggggagtgg ccacagcccc tcagcaggtg 2580

ctgtctaatg gcgtgcccgt gtccaacttc acctacagcc ccgacaccaa ggtgctggac 2640

atctgcgtgt cactgctgat gggagagcag tttctggtgt cctggtgctg a 2691

<210> 47

<211> 2691

<212> DNA

<213> Artificial sequence

<220>

<223> sp7+hGAAco2-Δ-47

<400> 47

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcccaaga 60

gctgtgccta cccaatgcga cgtgccaccc aactcccgat tcgactgcgc gccagataag 120

gctattaccc aagagcagtg tgaagccaga ggttgctgct acatcccagc gaagcaagga 180

ttgcaaggcg cccaaatggg acaaccttgg tgtttcttcc ccccttcgta cccatcatat 240

aaactcgaaa acctgtcctc ttcggaaatg ggttatactg ccaccctcac cagaactact 300

cctactttct tcccgaaaga catcttgacc ttgaggctgg acgtgatgat ggagactgaa 360

aaccggctgc atttcactat caaagatcct gccaatcggc gatacgaggt ccctctggaa 420

acccctcacg tgcactcacg ggctccttct ccgctttact ccgtcgaatt ctctgaggaa 480

cccttcggag tgatcgttag acgccagctg gatggtagag tgctgttgaa cactactgtg 540

gccccacttt tcttcgctga ccagtttctg caactgtcca cttccctgcc atcccagtac 600

attactggac tcgccgaaca cctgtcgcca ctgatgctct cgacctcttg gactagaatc 660

actttgtgga acagagactt ggcccctact ccggggagcaa atctgtacgg aagccaccct 720

ttttacctgg cgctcgaaga tggcggatcc gctcacggag tgttcctgct gaatagcaac 780

gcaatggacg tggtgctgca accttcccct gcactcagtt ggagaagtac cgggggtatt 840

ctggacgtgt acatcttcct cggaccagaa cccaagagcg tggtgcagca atatctggac 900

gtggtcggat acccttttat gcctccttac tggggactgg gattccacct ttgccgttgg 960

ggctactcat ccaccgccat taccagacag gtggtggaga atatgaccag agcccacttc 1020

cctctcgacg tgcagtggaa cgatctggac tatatggact cccggagaga tttcaccttc 1080

aacaaggacg ggttccgcga ttttcccgcg atggttcaag agctccacca gggtggtcga 1140

agatatatga tgatcgtcga cccagccatt tcgagcagcg gacccgctgg atctttataga 1200

ccttacgacg aaggccttag gagaggagtg ttcatcacaa acgagactgg acagcctttg 1260

atcggtaaag tgtggcctgg atcaaccgcc tttcctgact ttaccaatcc cactgccttg 1320

gcttggtggg aggacatggt ggccgaattc cacgaccaag tcccctttga tggaatgtgg 1380

atcgatatga acgaaccaag caattttatc agaggttccg aagacggttg ccccaacaac 1440

gaactggaaa accctcctta tgtgccccgga gtcgtgggcg gaacattaca ggccgcgact 1500

atttgcgcca gcagccacca attcctgtcc actcactaca acctccacaa cctttatgga 1560

ttaaccgaag ctattgcaag tcacagggct ctggtgaagg ctagagggac taggcccttt 1620

gtgatctccc gatccacctt tgccggacac gggagatacg ccggtcactg gactggtgac 1680

gtgtggagct catgggaaca actggcctcc tccgtgccgg aaatcttaca gttcaacctt 1740

ctgggtgtcc ctcttgtcgg agcagacgtg tgtgggtttc ttggtaacac ctccgaggaa 1800

ctgtgtgtgc gctggactca actgggtgca ttctacccat tcatgagaaa ccacaactcc 1860

ttgctgtccc tgccacaaga gccctactcg ttcagcgagc ctgcacaaca ggctatgcgg 1920

aaggcactga ccctgagata cgccctgctt ccacacttat acactctctt ccatcaagcg 1980

catgtggcag gagaaaccgt tgcaaggcct cttttccttg aattccccaa ggattcctcg 2040

acttggacgg tggatcatca gctgctgtgg ggagaagctc tgctgattac tccagtgttg 2100

caagccggaa aagctgaggt gaccggatac tttccgctgg gaacctggta cgacctccag 2160

actgtccctg ttgaagccct tggatcactg cctccgcctc cggcagctcc acgcgaacca 2220

gctatacatt ccgagggaca gtgggttaca ttaccagctc ctctggacac aatcaacgtc 2280

cacttaagag ctggctacat tatccctctg caaggaccag gactgactac gaccgagagc 2340

agacagcagc caatggcact ggctgtggct ctgaccaagg gaggggaagc tagaggagaa 2400

ctcttctggg atgatgggga gtcccttgaa gtgctggaaa gaggcgctta cactcaagtc 2460

attttccttg cacggaacaa caccattgtg aacgaattgg tgcgagtgac cagcgaagga 2520

gctggacttc aactgcagaa ggtcactgtg ctcggagtgg ctaccgctcc tcagcaagtg 2580

ctgtcgaatg gagtccccgt gtcaaacttt acctactccc ctgacactaa ggtgctcgac 2640

atttgcgtgt ccctcctgat gggagagcag ttccttgtgt cctggtgttg a 2691

<210> 48

<211> 2652

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco1-Δ-42

<400> 48

gcccaccccg gcagacctag agctgtgcct acccagtgtg acgtgccccc caacagcaga 60

ttcgactgcg cccctgacaa ggccatcacc caggaacagt gcgaggccag aggctgctgc 120

tacatccctg ccaagcaggg actgcagggc gctcagatgg gacagccctg gtgcttcttc 180

ccaccctcct accccagcta caagctggaa aacctgagca gcagcgagat gggctacacc 240

gccaccctga ccagaaccac ccccacattc ttcccaaagg acatcctgac cctgcggctg 300

gacgtgatga tggaaaccga gaaccggctg cacttcacca tcaaggaccc cgccaatcgg 360

agatacgagg tgcccctgga aaccccccac gtgcactcta gagcccccag ccctctgtac 420

agcgtggaat tcagcgagga acccttcggc gtgatcgtgc ggagacagct ggatggcaga 480

gtgctgctga acaccaccgt ggcccctctg ttcttcgccg accagttcct gcagctgagc 540

accagcctgc ccagccagta catcacagga ctggccgagc acctgagccc cctgatgctg 600

agcacatcct ggacccggat caccctgtgg aacagggatc tggcccctac ccctggcgcc 660

aatctgtacg gcagccaccc tttctacctg gccctggaag atggcggatc tgcccacgga 720

gtgtttctgc tgaactccaa cgccatggac gtggtgctgc agcctagccc tgccctgtct 780

tggagaagca caggcggcat cctggatgtg tacatctttc tgggccccga gcccaagagc 840

gtggtgcagc agtatctgga tgtcgtgggc taccccttca tgcccccctta ctggggcctg 900

ggattccacc tgtgcagatg gggctactcc agcaccgcca tcaccagaca ggtggtggaa 960

aacatgacca gagcccactt cccactggat gtgcagtgga acgacctgga ctacatggac 1020

agcagacggg acttcacctt caacaaggac ggcttccggg acttccccgc catggtgcag 1080

gaactgcatc agggcggcag acggtacatg atgatcgtgg atcccgccat cagctcctct 1140

ggccctgccg gctcttacag accctacgac gagggcctgc ggagaggcgt gttcatcacc 1200

aacgagacag gccagcccct gatcggcaaa gtgtggcctg gcagcacagc cttccccgac 1260

ttcaccaatc ctaccgccct ggcttggtgg gaggacatgg tggccgagtt ccacgaccag 1320

gtgcccttcg acggcatgtg gatcgacatg aacgagccca gcaacttcat ccggggcagc 1380

gaggatggct gccccaacaa cgaactggaa aatccccctt acgtgcccgg cgtcgtgggc 1440

ggaacactgc aggccgctac aatctgtgcc agcagccacc agtttctgag cacccactac 1500

aacctgcaca acctgtacgg cctgaccgag gccattgcca gccaccgcgc tctcgtgaaa 1560

gccagaggca cacggccctt cgtgatcagc agaagcacct ttgccggcca cggcagatac 1620

gccggacatt ggactggcga cgtgtggtcc tcttggggagc agctggcctc tagcgtgccc 1680

gagatcctgc agttcaatct gctgggcgtg ccactcgtgg gcgccgatgt gtgtggcttc 1740

ctgggcaaca cctccgagga actgtgtgtg cggtggacac agctgggcgc cttctaccct 1800

ttcatgagaa accacaacag cctgctgagc ctgccccagg aaccctacag ctttagcgag 1860

cctgcacagc aggccatgcg gaaggccctg acactgagat acgctctgct gccccacctg 1920

tacaccctgt ttcaccaggc ccatgtggcc ggcgagacag tggccagacc tctgtttctg 1980

gaattcccca aggacagcag cacctggacc gtggaccatc agctgctgtg gggagaggct 2040

ctgctgatta ccccagtgct gcaggcaggc aaggccgaag tgaccggcta ctttcccctg 2100

ggcacttggt acgacctgca gaccgtgcct gtggaagccc tgggatctct gcctccacct 2160

cctgccgctc ctagagagcc tgccattcac tctgagggcc agtgggtcac actgcctgcc 2220

cccctggata ccatcaacgt gcacctgagg gccggctaca tcataccact gcagggacct 2280

ggcctgacca ccaccgagtc tagacagcag ccaatggccc tggccgtggc cctgaccaaa 2340

ggcggagaag ctaggggcga gctgttctgg gacgatggcg agagcctgga agtgctggaa 2400

agaggcgcct atacccaagt gatcttcctg gcccggaaca acaccatcgt gaacgagctg 2460

gtgcgcgtga cctctgaagg cgctggactg cagctgcaga aagtgaccgt gctggggagtg 2520

gccacagccc ctcagcaggt gctgtctaat ggcgtgcccg tgtccaactt cacctacagc 2580

cccgacacca aggtgctgga catctgcgtg tcactgctga tgggagagca gtttctggtg 2640

tcctggtgct ga 2652

<210> 49

<211> 2652

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco2-Δ-42

<400> 49

gcccatcctg gtagaccaag agctgtgcct acccaatgcg acgtgccacc caactcccga 60

ttcgactgcg cgccagataa ggctattacc caagagcagt gtgaagccag aggttgctgc 120

tacatcccag cgaagcaagg attgcaaggc gcccaaatgg gacaaccttg gtgtttcttc 180

cccccttcgt acccatcata taaactcgaa aacctgtcct cttcggaaat gggttatact 240

gccaccctca ccagaactac tcctactttc ttcccgaaag acatcttgac cttgaggctg 300

gacgtgatga tggagactga aaaccggctg catttcacta tcaaagatcc tgccaatcgg 360

cgatacgagg tccctctgga aacccctcac gtgcactcac gggctccttc tccgctttac 420

tccgtcgaat tctctgagga acccttcgga gtgatcgtta gacgccagct ggatggtaga 480

gtgctgttga acactactgt ggccccactt ttcttcgctg accagtttct gcaactgtcc 540

acttccctgc catcccagta cattactgga ctcgccgaac acctgtcgcc actgatgctc 600

tcgacctctt ggactagaat cactttgtgg aacagagact tggcccctac tccggggagca 660

aatctgtacg gaagccaccc tttttacctg gcgctcgaag atggcggatc cgctcacgga 720

gtgttcctgc tgaatagcaa cgcaatggac gtggtgctgc aaccttcccc tgcactcagt 780

tggagaagta ccgggggtat tctggacgtg tacatcttcc tcggaccaga acccaagagc 840

gtggtgcagc aatatctgga cgtggtcgga taccctttta tgcctcctta ctggggactg 900

ggattccacc tttgccgttg gggctactca tccaccgcca ttaccagaca ggtggtggag 960

aatatgacca gagcccactt ccctctcgac gtgcagtgga acgatctgga ctatatggac 1020

tcccggagag atttcacctt caacaaggac gggttccgcg attttcccgc gatggttcaa 1080

gagctccacc agggtggtcg aagatatatg atgatcgtcg acccagccat ttcgagcagc 1140

ggacccgctg gatcttatag accttacgac gaaggcctta ggagaggagt gttcatcaca 1200

aacgagactg gacagccttt gatcggtaaa gtgtggcctg gatcaaccgc ctttcctgac 1260

tttaccaatc ccactgcctt ggcttggtgg gaggacatgg tggccgaatt ccacgaccaa 1320

gtcccctttg atggaatgtg gatcgatatg aacgaaccaa gcaattttat cagaggttcc 1380

gaagacggtt gccccaacaa cgaactggaa aaccctcctt atgtgcccgg agtcgtgggc 1440

ggaacattac aggccgcgac tatttgcgcc agcagccacc aattcctgtc cactcactac 1500

aacctccaca acctttatgg attaaccgaa gctattgcaa gtcacagggc tctggtgaag 1560

gctagaggga ctaggccctt tgtgatctcc cgatccacct ttgccggaca cgggatac 1620

gccggtcact ggactggtga cgtgtggagc tcatgggaac aactggcctc ctccgtgccg 1680

gaaatcttac agttcaacct tctgggtgtc cctcttgtcg gagcagacgt gtgtgggttt 1740

cttggtaaca cctccgagga actgtgtgtg cgctggactc aactgggtgc attctaccca 1800

ttcatgagaa accacaactc cttgctgtcc ctgccacaag agccctactc gttcagcgag 1860

cctgcacaac aggctatgcg gaaggcactg accctgagat acgccctgct tccacactta 1920

tacactctct tccatcaagc gcatgtggca ggagaaaccg ttgcaaggcc tcttttcctt 1980

gaattcccca aggattcctc gacttggacg gtggatcatc agctgctgtg gggagaagct 2040

ctgctgatta ctccagtgtt gcaagccgga aaagctgagg tgaccggata ctttccgctg 2100

ggaacctggt acgacctcca gactgtccct gttgaagccc ttggatcact gcctccgcct 2160

ccggcagctc cacgcgaacc agctatacat tccgagggac agtgggttac attaccagct 2220

cctctggaca caatcaacgt ccacttaaga gctggctaca ttatccctct gcaaggacca 2280

ggactgacta cgaccgagag cagacagcag ccaatggcac tggctgtggc tctgaccaag 2340

ggaggggaag ctagaggaga actcttctgg gatgatgggg agtcccttga agtgctggaa 2400

agaggcgctt acactcaagt cattttcctt gcacggaaca acaccattgt gaacgaattg 2460

gtgcgagtga ccagcgaagg agctggactt caactgcaga aggtcactgt gctcggagtg 2520

gctaccgctc ctcagcaagt gctgtcgaat ggagtccccg tgtcaaactt tacctactcc 2580

cctgacacta aggtgctcga catttgcgtg tccctcctga tgggagagca gttccttgtg 2640

tcctggtgtt ga 2652

<210> 50

<211> 2649

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco1-Δ-43

<400> 50

caccccggca gacctagagc tgtgcctacc cagtgtgacg tgcccccccaa cagcagattc 60

gactgcgccc ctgacaaggc catcacccag gaacagtgcg aggccagagg ctgctgctac 120

atccctgcca agcagggact gcagggcgct cagatgggac agccctggtg cttcttccca 180

ccctcctacc ccagctacaa gctggaaaac ctgagcagca gcgagatggg ctacaccgcc 240

accctgacca gaaccacccc cacattcttc ccaaaggaca tcctgaccct gcggctggac 300

gtgatgatgg aaaccgagaa ccggctgcac ttcaccatca aggaccccgc caatcggaga 360

tacgaggtgc ccctggaaac cccccacgtg cactctagag cccccagccc tctgtacagc 420

gtggaattca gcgaggaacc cttcggcgtg atcgtgcgga gacagctgga tggcagagtg 480

ctgctgaaca ccaccgtggc ccctctgttc ttcgccgacc agttcctgca gctgagcacc 540

agcctgccca gccagtacat cacaggactg gccgagcacc tgagccccct gatgctgagc 600

acatcctgga cccggatcac cctgtggaac agggatctgg cccctacccc tggcgccaat 660

ctgtacggca gccacccttt ctacctggcc ctggaagatg gcggatctgc ccacggagtg 720

tttctgctga actccaacgc catggacgtg gtgctgcagc ctagccctgc cctgtcttgg 780

agaagcacag gcggcatcct ggatgtgtac atctttctgg gccccgagcc caagagcgtg 840

gtgcagcagt atctggatgt cgtgggctac cccttcatgc ccccttactg gggcctggga 900

ttccacctgt gcagatgggg ctactccagc accgccatca ccagacaggt ggtggaaaac 960

atgaccagag cccacttccc actggatgtg cagtggaacg acctggacta catggacagc 1020

agacgggact tcaccttcaa caaggacggc ttccgggact tccccgccat ggtgcaggaa 1080

ctgcatcagg gcggcagacg gtacatgatg atcgtggatc ccgccatcag ctcctctggc 1140

cctgccggct cttacagacc ctacgacgag ggcctgcgga gaggcgtgtt catcaccaac 1200

gagacaggcc agcccctgat cggcaaagtg tggcctggca gcacagcctt ccccgacttc 1260

accaatccta ccgccctggc ttggtgggag gacatggtgg ccgagttcca cgaccaggtg 1320

cccttcgacg gcatgtggat cgacatgaac gagcccagca acttcatccg gggcagcgag 1380

gatggctgcc ccaacaacga actggaaaat cccccttacg tgcccggcgt cgtgggcgga 1440

acactgcagg ccgctacaat ctgtgccagc agccaccagt ttctgagcac ccactacaac 1500

ctgcacaacc tgtacggcct gaccgaggcc attgccagcc accgcgctct cgtgaaagcc 1560

agaggcacac ggcccttcgt gatcagcaga agcacctttg ccggccacgg cagatacgcc 1620

ggacattgga ctggcgacgt gtggtcctct tgggagcagc tggcctctag cgtgcccgag 1680

atcctgcagt tcaatctgct gggcgtgcca ctcgtgggcg ccgatgtgtg tggcttcctg 1740

ggcaacacct ccgaggaact gtgtgtgcgg tggacacagc tgggcgcctt ctaccctttc 1800

atgagaaacc acaacagcct gctgagcctg ccccaggaac cctacagctt tagcgagcct 1860

gcacagcagg ccatgcggaa ggccctgaca ctgagatacg ctctgctgcc ccacctgtac 1920

accctgtttc accaggccca tgtggccggc gagacagtgg ccagacctct gtttctggaa 1980

ttccccaagg acagcagcac ctggaccgtg gaccatcagc tgctgtgggg agaggctctg 2040

ctgattaccc cagtgctgca ggcaggcaag gccgaagtga ccggctactt tcccctgggc 2100

acttggtacg acctgcagac cgtgcctgtg gaagccctgg gatctctgcc tccacctcct 2160

gccgctccta gagagcctgc cattcactct gagggccagt gggtcacact gcctgccccc 2220

ctggatacca tcaacgtgca cctgagggcc ggctacatca taccactgca gggacctggc 2280

ctgaccacca ccgagtctag acagcagcca atggccctgg ccgtggccct gaccaaaggc 2340

ggagaagcta ggggcgagct gttctgggac gatggcgaga gcctggaagt gctggaaaga 2400

ggcgcctata cccaagtgat cttcctggcc cggaacaaca ccatcgtgaa cgagctggtg 2460

cgcgtgacct ctgaaggcgc tggactgcag ctgcagaaag tgaccgtgct gggagtggcc 2520

acagcccctc agcaggtgct gtctaatggc gtgcccgtgt ccaacttcac ctacagcccc 2580

gacaccaagg tgctggacat ctgcgtgtca ctgctgatgg gagagcagtt tctggtgtcc 2640

tggtgctga 2649

<210> 51

<211> 2649

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco2-Δ-43

<400> 51

catcctggta gaccaagagc tgtgcctacc caatgcgacg tgccacccaa ctcccgattc 60

gactgcgcgc cagataaggc tattacccaa gagcagtgtg aagccagagg ttgctgctac 120

atcccagcga agcaaggatt gcaaggcgcc caaatgggac aaccttggtg tttcttcccc 180

ccttcgtacc catcatataa actcgaaaac ctgtcctctt cggaaatggg ttatactgcc 240

accctcacca gaactactcc tactttcttc ccgaaagaca tcttgacctt gaggctggac 300

gtgatgatgg agactgaaaa ccggctgcat ttcactatca aagatcctgc caatcggcga 360

tacgaggtcc ctctggaaac ccctcacgtg cactcacggg ctccttctcc gctttatcc 420

gtcgaattct ctgaggaacc cttcggagtg atcgttagac gccagctgga tggtagagtg 480

ctgttgaaca ctactgtggc cccacttttc ttcgctgacc agtttctgca actgtccact 540

tccctgccat cccagtacat tactggactc gccgaacacc tgtcgccact gatgctctcg 600

acctcttgga ctagaatcac tttgtggaac agagacttgg cccctactcc gggagcaaat 660

ctgtacggaa gccacccttt ttacctggcg ctcgaagatg gcggatccgc tcacggagtg 720

ttcctgctga atagcaacgc aatggacgtg gtgctgcaac cttcccctgc actcagttgg 780

agaagtaccg ggggtattct ggacgtgtac atcttcctcg gaccagaacc caagagcgtg 840

gtgcagcaat atctggacgt ggtcggatac ccttttatgc ctccttatactg gggactggga 900

ttccaccttt gccgttgggg ctactcatcc accgccatta ccagacaggt ggtggagaat 960

atgaccagag cccacttccc tctcgacgtg cagtggaacg atctggacta tatggactcc 1020

cggagagatt tcaccttcaa caaggacggg ttccgcgatt ttcccgcgat ggttcaagag 1080

ctccaccagg gtggtcgaag atatatgatg atcgtcgacc cagccatttc gagcagcgga 1140

cccgctggat cttatagacc ttacgacgaa ggccttagga gaggagtgtt catcacaaac 1200

gagactggac agcctttgat cggtaaagtg tggcctggat caaccgcctt tcctgacttt 1260

accaatccca ctgccttggc ttggtgggag gacatggtgg ccgaattcca cgaccaagtc 1320

ccctttgatg gaatgtggat cgatatgaac gaaccaagca attttatcag aggttccgaa 1380

gacggttgcc ccaacaacga actggaaaac cctccttatg tgcccggagt cgtgggcgga 1440

acattacagg ccgcgactat ttgcgccagc agccaccaat tcctgtccac tcactacaac 1500

ctccacaacc tttatggatt aaccgaagct attgcaagtc acagggctct ggtgaaggct 1560

agagggacta ggccctttgt gatctcccga tccacctttg ccggacacgg gagatacgcc 1620

ggtcactgga ctggtgacgt gtggagctca tgggaacaac tggcctcctc cgtgccggaa 1680

atcttacagt tcaaccttct gggtgtcct cttgtcggag cagacgtgtg tgggtttctt 1740

ggtaacacct ccgaggaact gtgtgtgcgc tggactcaac tgggtgcatt ctacccattc 1800

atgagaaacc acaactcctt gctgtccctg ccacaagagc cctactcgtt cagcgagcct 1860

gcacaacagg ctatgcggaa ggcactgacc ctgagatacg ccctgcttcc acacttatac 1920

actctcttcc atcaagcgca tgtggcagga gaaaccgttg caaggcctct tttccttgaa 1980

ttccccaagg attcctcgac ttggacggtg gatcatcagc tgctgtgggg agaagctctg 2040

ctgattactc cagtgttgca agccggaaaa gctgaggtga ccggatactt tccgctggga 2100

acctggtacg acctccagac tgtccctgtt gaagcccttg gatcactgcc tccgcctccg 2160

gcagctccac gcgaaccagc tatacattcc gagggacagt gggttacatt accagctcct 2220

ctggacacaa tcaacgtcca cttaagagct ggctacatta tccctctgca aggaccagga 2280

ctgactacga ccgagagcag acagcagcca atggcactgg ctgtggctct gaccaaggga 2340

ggggaagcta gaggagaact cttctggggat gatggggagt cccttgaagt gctggaaaga 2400

ggcgcttaca ctcaagtcat tttccttgca cggaacaaca ccattgtgaa cgaattggtg 2460

cgagtgacca gcgaaggagc tggacttcaa ctgcagaagg tcactgtgct cggagtggct 2520

accgctcctc agcaagtgct gtcgaatgga gtccccgtgt caaactttac ctactcccct 2580

gacactaagg tgctcgacat ttgcgtgtcc ctcctgatgg gagagcagtt ccttgtgtcc 2640

tggtgttga 2649

<210> 52

<211> 2637

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco1-Δ-47

<400> 52

cctagagctg tgcctaccca gtgtgacgtg ccccccaaca gcagattcga ctgcgcccct 60

gacaaggcca tcacccagga acagtgcgag gccagaggct gctgctacat ccctgccaag 120

cagggactgc agggcgctca gatggggacag ccctggtgct tcttcccacc ctcctacccc 180

agctacaagc tggaaaacct gagcagcagc gagatgggct acaccgccac cctgaccaga 240

accacccccca cattcttccc aaaggacatc ctgaccctgc ggctggacgt gatgatggaa 300

accgagaacc ggctgcactt caccatcaag gaccccgcca atcggagata cgaggtgccc 360

ctggaaaccc cccacgtgca ctctagagcc cccagccctc tgtacagcgt ggaattcagc 420

gaggaaccct tcggcgtgat cgtgcggaga cagctggatg gcagagtgct gctgaacacc 480

accgtggccc ctctgttctt cgccgaccag ttcctgcagc tgagcaccag cctgcccagc 540

cagtacatca caggactggc cgagcacctg agccccctga tgctgagcac atcctggacc 600

cggatcaccc tgtggaacag ggatctggcc cctacccctg gcgccaatct gtacggcagc 660

caccctttct acctggccct ggaagatggc ggatctgccc acggagtgtt tctgctgaac 720

tccaacgcca tggacgtggt gctgcagcct agccctgccc tgtcttggag aagcacaggc 780

ggcatcctgg atgtgtacat ctttctgggc cccgagccca agagcgtggt gcagcagtat 840

ctggatgtcg tgggctaccc cttcatgccc ccttactggg gcctgggatt ccacctgtgc 900

agatggggct actccagcac cgccatcacc agacaggtgg tggaaaacat gaccagagcc 960

cacttcccac tggatgtgca gtggaacgac ctggactaca tggacagcag acgggacttc 1020

accttcaaca aggacggctt ccgggacttc cccgccatgg tgcaggaact gcatcagggc 1080

ggcagacggt acatgatgat cgtggatccc gccatcagct cctctggccc tgccggctct 1140

tacagaccct acgacgaggg cctgcggaga ggcgtgttca tcaccaacga gacaggccag 1200

cccctgatcg gcaaagtgtg gcctggcagc acagccttcc ccgacttcac caatcctacc 1260

gccctggctt ggtggggagga catggtggcc gagttccacg accaggtgcc cttcgacggc 1320

atgtggatcg acatgaacga gcccagcaac ttcatccggg gcagcgagga tggctgcccc 1380

aacaacgaac tggaaaatcc cccttacgtg cccggcgtcg tgggcggaac actgcaggcc 1440

gctacaatct gtgccagcag ccaccagttt ctgagcaccc actacaacct gcacaacctg 1500

tacggcctga ccgaggccat tgccagccac cgcgctctcg tgaaagccag aggcacacgg 1560

cccttcgtga tcagcagaag cacctttgcc ggccacggca gatacgccgg acattggact 1620

ggcgacgtgt ggtcctcttg ggagcagctg gcctctagcg tgcccgagat cctgcagttc 1680

aatctgctgg gcgtgccact cgtgggcgcc gatgtgtgtg gcttcctggg caacacctcc 1740

gaggaactgt gtgtgcggtg gacacagctg ggcgccttct accctttcat gagaaaccac 1800

aacagcctgc tgagcctgcc ccaggaaccc tacagcttta gcgagcctgc acagcaggcc 1860

atgcggaagg ccctgacact gagatacgct ctgctgcccc acctgtacac cctgtttcac 1920

caggcccatg tggccggcga gacagtggcc agacctctgt ttctggaatt ccccaaggac 1980

agcagcacct ggaccgtgga ccatcagctg ctgtggggag aggctctgct gattacccca 2040

gtgctgcagg caggcaaggc cgaagtgacc ggctactttc ccctgggcac ttggtacgac 2100

ctgcagaccg tgcctgtgga agccctggga tctctgcctc cacctcctgc cgctcctaga 2160

gagcctgcca ttcactctga gggccagtgg gtcacactgc ctgcccccct ggataccatc 2220

aacgtgcacc tgagggccgg ctacatcata ccactgcagg gacctggcct gaccaccacc 2280

gagtctagac agcagccaat ggccctggcc gtggccctga ccaaaggcgg agaagctagg 2340

ggcgagctgt tctgggacga tggcgagagc ctggaagtgc tggaaagagg cgcctatacc 2400

caagtgatct tcctggcccg gaacaacacc atcgtgaacg agctggtgcg cgtgacctct 2460

gaaggcgctg gactgcagct gcagaaagtg accgtgctgg gagtggccac agcccctcag 2520

caggtgctgt ctaatggcgt gcccgtgtcc aacttcacct acagccccga caccaaggtg 2580

ctggacatct gcgtgtcact gctgatggga gagcagtttc tggtgtcctg gtgctga 2637

<210> 53

<211> 2637

<212> DNA

<213> Artificial sequence

<220>

<223> hGAAco2-Δ-47

<400> 53

ccaagagctg tgcctaccca atgcgacgtg ccacccaact cccgattcga ctgcgcgcca 60

gataaggcta ttacccaaga gcagtgtgaa gccagaggtt gctgctacat cccagcgaag 120

caaggattgc aaggcgccca aatgggacaa ccttggtgtt tcttcccccc ttcgtaccca 180

tcatataaac tcgaaaacct gtcctcttcg gaaatgggtt atactgccac cctcaccaga 240

actactccta ctttcttccc gaaagacatc ttgaccttga ggctggacgt gatgatggag 300

actgaaaacc ggctgcattt cactatcaaa gatcctgcca atcggcgata cgaggtccct 360

ctggaaaccc ctcacgtgca ctcacgggct ccttctccgc tttactccgt cgaattctct 420

gaggaaccct tcggagtgat cgttagacgc cagctggatg gtagagtgct gttgaacact 480

actgtggccc cacttttctt cgctgaccag tttctgcaac tgtccacttc cctgccatcc 540

cagtacatta ctggactcgc cgaacacctg tcgccactga tgctctcgac ctcttggact 600

agaatcactt tgtggaacag agacttggcc cctactccgg gagcaaatct gtacggaagc 660

cacccttttt acctggcgct cgaagatggc ggatccgctc acggagtgtt cctgctgaat 720

agcaacgcaa tggacgtggt gctgcaacct tcccctgcac tcagttggag aagtaccggg 780

ggtattctgg acgtgtacat cttcctcgga ccagaaccca agagcgtggt gcagcaatat 840

ctggacgtgg tcggataccc ttttatgcct ccttactggg gactgggatt ccacctttgc 900

cgttggggct actcatccac cgccattacc agacaggtgg tggagaatat gaccagagcc 960

cacttccctc tcgacgtgca gtggaacgat ctggactata tggactcccg gagagatttc 1020

accttcaaca aggacgggtt ccgcgatttt cccgcgatgg ttcaagagct ccaccagggt 1080

ggtcgaagat atatgatgat cgtcgaccca gccatttcga gcagcggacc cgctggatct 1140

tatagacctt acgacgaagg ccttaggaga ggagtgttca tcacaaacga gactggacag 1200

cctttgatcg gtaaagtgtg gcctggatca accgcctttc ctgactttac caatcccact 1260

gccttggctt ggtggggagga catggtggcc gaattccacg accaagtccc ctttgatgga 1320

atgtggatcg atatgaacga accaagcaat tttatcagag gttccgaaga cggttgcccc 1380

aacaacgaac tggaaaaccc tccttatgtg cccggagtcg tgggcggaac attacaggcc 1440

gcgactattt gcgccagcag ccaccaattc ctgtccactc actacaacct ccacaacctt 1500

tatggattaa ccgaagctat tgcaagtcac agggctctgg tgaaggctag agggactagg 1560

ccctttgtga tctcccgatc cacctttgcc ggacacggga gatacgccgg tcactggact 1620

ggtgacgtgt ggagctcatg ggaacaactg gcctcctccg tgccggaaat cttacagttc 1680

aaccttctgg gtgtccctct tgtcggagca gacgtgtgtg ggtttcttgg taacacctcc 1740

gaggaactgt gtgtgcgctg gactcaactg ggtgcattct acccattcat gagaaaccac 1800

aactccttgc tgtccctgcc acaagagccc tactcgttca gcgagcctgc acaacaggct 1860

atgcggaagg cactgaccct gagatacgcc ctgcttccac acttatacac tctcttccat 1920

caagcgcatg tggcaggaga aaccgttgca aggcctcttt tccttgaatt ccccaaggat 1980

tcctcgactt ggacggtgga tcatcagctg ctgtggggag aagctctgct gattactcca 2040

gtgttgcaag ccggaaaagc tgaggtgacc ggatactttc cgctgggaac ctggtacgac 2100

ctccagactg tccctgttga agcccttgga tcactgcctc cgcctccggc agctccacgc 2160

gaaccagcta tacattccga gggacagtgg gttacattac cagctcctct ggacacaatc 2220

aacgtccact taagagctgg ctacattatc cctctgcaag gaccaggact gactacgacc 2280

gagagcagac agcagccaat ggcactggct gtggctctga ccaagggagg ggaagctaga 2340

ggagaactct tctggggatga tggggagtcc cttgaagtgc tggaaagagg cgcttacact 2400

caagtcattt tccttgcacg gaacaacacc attgtgaacg aattggtgcg agtgaccagc 2460

gaaggagctg gacttcaact gcagaaggtc actgtgctcg gagtggctac cgctcctcag 2520

caagtgctgt cgaatggagt ccccgtgtca aactttacct actcccctga cactaaggtg 2580

ctcgacattt gcgtgtccct cctgatggga gagcagttcc ttgtgtcctg gtgttga 2637

Claims (33)

1. A recombinant AAV vector comprising an expression cassette comprising a nucleic acid molecule encoding a truncated GAA polypeptide that has 8 deleted at its N-terminal end compared with the parent GAA polypeptide. to 43 consecutive amino acids, wherein the parent GAA polypeptide corresponds to a precursor form of a GAA polypeptide that does not contain a signal peptide, and Wherein the truncated GAA polypeptide further comprises a signal peptide fused to its N-terminal end. 2. The recombinant AAV vector of claim 1, wherein the truncated GAA polypeptide has 8, 9, 10, 40, 41, 42, or 43 consecutive deletions at its N-terminal end compared to the parent GAA polypeptide. Amino acids. 3. The recombinant AAV vector of claim 1 or 2, wherein the truncated GAA polypeptide has 8, 42 or 43 consecutive amino acids truncated at its N-terminal end compared to the parent GAA polypeptide. 4. The recombinant AAV vector of claim 1 or 2, wherein the parent GAA polypeptide is human GAA. 5. The recombinant AAV vector of claim 1 or 2, wherein the parent GAA polypeptide is human GAA consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 33. 6. The recombinant AAV vector of claim 1 or 2, wherein the parent GAA polypeptide is human GAA consisting of the amino acid sequence of SEQ ID NO: 1. 7. The recombinant AAV vector of claim 1 or 2, wherein the truncated GAA polypeptide consists of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35. 8. The recombinant AAV vector of claim 1 or 2, wherein the fused signal peptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 of amino acid sequence. 9. The recombinant AAV vector of claim 1 or 2, wherein the fused signal peptide consists of the amino acid sequence of SEQ ID NO: 3. 10. The recombinant AAV vector of claim 1 or 2, wherein the nucleic acid molecule encoding a truncated GAA polypeptide is optimized to increase expression of the truncated GAA polypeptide in vivo and/or to increase response to the truncated GAA. Immune tolerance of polypeptides, the nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 composition. 11. The recombinant AAV vector of claim 1 or 2, wherein said nucleic acid molecule is operably linked to a promoter. 12. The recombinant AAV vector of claim 11, wherein said promoter is a liver-specific promoter. 13. The recombinant AAV vector of claim 12, wherein the promoter is selected from the group consisting of alpha-1 antitrypsin promoter, transthyretin promoter, albumin promoter and thyroxine-binding globulin promoter. 14. The recombinant AAV vector of claim 1 or 2, wherein the expression cassette further comprises an intron. 15. The recombinant AAV vector of claim 14, wherein the intron is selected from the group consisting of human β-globin b2 intron, FIX intron, chicken β-globin intron and SV40 intron. 16. The recombinant AAV vector of claim 14, wherein the intron is the modified human beta globin b2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, or the modified FIX intron of SEQ ID NO: 17 :21 modified chicken β-globin intron. 17. The recombinant AAV vector of claim 1 or 2, wherein the expression cassette comprises in the following order: an enhancer; a promoter, an intron; the nucleic acid molecule encoding the truncated GAA polypeptide; and polyadenosine signal. 18. The recombinant AAV vector of claim 1 or 2, wherein said expression cassette comprises in the following order: ApoE control region; hAAT promoter; human beta globin b2 intron; said truncated GAA polypeptide encoding Nucleic acid molecules; and bovine growth hormone polyadenylation signals. 19. The recombinant AAV vector of claim 1 or 2, wherein the expression cassette consists of the nucleotide sequence of any one of SEQ ID NO: 22 to 26. 20. The recombinant AAV vector of claim 1 or 2, wherein the recombinant AAV vector is a single-stranded or double-stranded self-complementary AAV vector. 21. The recombinant AAV vector of claim 1 or 2, wherein the recombinant AAV vector is a vector having a gene selected from the group consisting of AAV1, AAV2, variant AAV2, AAV3, variant AAV3, AAV3B, variant AAV3B, AAV4, AAV5, AAV6, variant AAV capsids of AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8 and porcine AAV or AAV vectors with chimeric capsids. 22. The recombinant AAV vector of claim 1 or 2, wherein the recombinant AAV vector has an AAV8, AAV9, AAVrh74 or AAV2i8 capsid. 23. The recombinant AAV vector of claim 1 or 2, wherein the recombinant AAV vector has an AAV8, AAV9 or AAVrh74 capsid. 24. The recombinant AAV vector of claim 1 or 2, wherein the recombinant AAV vector has an AAV8 capsid. 25. The recombinant AAV vector of claim 1 or 2, comprising an expression cassette and a modified capsid, the expression cassette comprising a nucleic acid molecule encoding a truncated GAA polypeptide consisting of SEQ ID NO: 27 The amino acid sequence composition, the truncated GAA polypeptide also includes a signal peptide fused to its N-terminal end, the signal peptide consists of the amino acid sequence of SEQ ID NO: 3, and the modified capsid includes AAVrh74 derived from or one or more variant VP capsid proteins selected from RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6. 26. A cell transformed with the recombinant AAV vector of any one of claims 1 to 25. 27. The cell of claim 26, wherein said cell is a hepatocyte or a muscle cell. 28. A pharmaceutical composition comprising the recombinant AAV vector of any one of claims 1 to 25 or the cell of claim 26 or 27 in a pharmaceutically acceptable carrier. 29. Use of the recombinant AAV vector of any one of claims 1 to 25, the cell of claim 26 or 27 or the pharmaceutical composition of claim 28 for the preparation of a medicament for the treatment of GAA deficiency or glycogen storage sick. 30. The use of claim 29, wherein the glycogen storage disease is selected from the group consisting of GSDI, GSDII, GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage diseases of the heart. 31. The use of claim 30, wherein the glycogen storage disease is GSDII or GSDIII. 32. The use of claim 31, wherein said glycogen storage disease is GSDIII. 33. Use of the recombinant AAV vector of any one of claims 1 to 25, the cell of claim 26 or 27 or the pharmaceutical composition of claim 28 for the preparation of a medicament for the treatment of glycogen storage disease while simultaneously The immunogenicity of the truncated GAA polypeptide is reduced.